Complement component c5 antibodies

ABSTRACT

The present disclosure relates to antibodies and polynucleotides encoding the same, that may be used to prevent, control, or reduce the activity of the complement pathway. In addition, the disclosure is directed to compositions and methods for diagnosing and treating diseases mediated by or involving complement C5. Specifically, the disclosure is related to C5 antibodies.

CROSS-REFERENCE

This application is a Divisional of U.S. patent application Ser. No.14/626,514, filed Feb. 19, 2015, which claims priority under 35 U.S.C.§119(e) from U.S. Provisional Application Ser. No. 61/768,374, filedFeb. 20, 2014, and U.S. Provisional Application Ser. No. 61/944,943,filed Feb. 26, 2014, all of which is hereby incorporated by reference intheir entirety.

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates to antibodies and compositions thereof,polynucleotides encoding the same, expression vectors and host cells forproduction of the antibodies, and compositions and methods fordiagnosing and treating diseases mediated by complement.

BACKGROUND OF THE INVENTION

The complement system is composed of nearly 50 individual proteins thatfunctions as a part of the innate immune system providing the initialphase of host defense, opsonization of foreign material, and tissuehomeostasis. (Ricklin D., 2010, Complement: a Key system for immunesurveillance and homeostasis. Nature: Immunology, 785-795) Thecomplement system is found in all multicellular organism andphylogenetically predates the formation of the adaptive immune system(Zarkadis I. K., 2001 Phylogenetic aspects of the complement system.Development and Comparative Immunology, 745-762.). Activation of thecomplement system occurs along three primary pathways: classical, lectinand alternative pathways. FIG. 1 shows a schematic representation of thethree primary complement pathways. See also, Donoso, et al., “The Roleof Inflammation in the Pathogenesis of Age-related MacularDegeneration”, Survey of Ophthalmology, Vol. 51, No. 2, March-April2006.

During the activation process sequential protein-protein interactionsand proteolytic activity leads to the generation of the C3 and C5convertases. These convertases are responsible for producing complementactivation split products that represent the effector molecules of thecomplement cascade important for opsonization, generation ofanaphylatoxins, and the formation of the membrane attack complex (MAC).The latter of these is essential for the lytic activity of thecomplement cascade (Ricklin D., 2010). Under normal conditionsactivation of the complement cascades provides defense againstpathogenic bacterial, viruses as well as clearance of diseased andinjured tissue. Normally, the formation of MAC does not affectsurrounding tissue due to the presence of cell surface and solubleregulatory components which include CFH, CFH related proteins, C4BP,CD46, CD55, CD59 and complement factor I (CFI). However, when excessactivation occurs or when there is a failure in complement regulatorycomponents, both acute and chronic disease states are induced. Examplesin which uncontrolled complement activation is recognized as causativeto human pathologies include: Glomerulonephritis, Systemic LupusErythematosus, Paroxysmal Nocturnal Hemoglobinuria, Alzheimer's,Hereditary Angioedema, Myasthenia Gravis and Age-related MacularDegeneration (AMD) (Ricklin & Lambris, 2013, Complement in Immune andinflammatory Disorders: Pthaological Mechanisms. Journal of Immunology,3831-3838).

C5 is a 190 kDa protein comprising two polypeptide chains (α, 115 kDaand β, 75 kDa) that are linked together by disulfide bonds. C5convertase cleaves at an arginine residue 75 amino acids downstream fromthe C5 α-chain N terminus generating the 7.4 Kd C5a and 180 Kd C5bcomplement split products. The C5b component serves as the initiationcomponent for the assembly of the membrane attack complex (MAC) throughthe sequential addition of C6, C7, C8 and C9. The C6-C8 subunitsassemble in a 1:1 relationship to C5b while multiple C9 subunits areincorporate into the complex generating a non-specific pore in bothprokaryotic and eukaryotic plasma membranes FIG. 2. See also, Bubeck D.,2014, “The making of a macromolecular machine: assembly of the membraneattack complex” Biochemistry, 53(12):1908-15. The formation of MAC onthe cell surface has several consequences for the cells. At high levelsthe unregulated influx and efflux of solutes leads to cellular swellingand eventual cell lysis. This causes the uncontrolled release ofcellular material promoting a pro-inflammatory environment and cellularloss. Formation of MAC at sublytic concentrations on the cell surfacecan contribute to release of pro-inflammatory and pro-agniogeniccytokines and growth factors, elevation in cellular stress and eventualnecrotic cell death.

Age-related Macular Degeneration (AMD) is the leading cause of blindnessin the elderly developed countries. In the US population alone theprevalence of advanced forms of AMD associated with vision loss occursin nearly 2 million individuals. Another 7 million individuals withintermediate AMD are at a high risk for development of advanced forms ofAMD. Inclusion of the European population nearly doubles the number ofimpacted individuals. AMD is characterized by a progressive loss ofvision attributable to a para-inflammatory process causing theprogressive degeneration of the neuroretina, and support tissues whichinclude the retinal pigmented epithelium (RPE) and choriocapillaris. Themajority of clinically significant vision loss occurs when theneurodegenerative changes impact the center of the retina within ahighly specialized region of the eye responsible for fine visual acuity,the macula. The disease has a tremendous impact on the physical andmental health of the individual due to vision loss and increaseddependence on family members to perform everyday tasks.

The deregulation of the complement system is highly correlated with thedevelopment of AMD. First, genetic mutations in over 20 genes have beencorrelated with a person's risk of developing AMD, accounting for anestimated 70% of total risk. (Fritsche et al., “Age related MacularDegeneration: Genetics and Biology Coming Together”, Annu Rev GenomicsHum Genet. 2014; 15:151-71). Within these 20 genes, five are complementgenes, which alone account for 57% of total risk in the development ofthe advanced forms of AMD. In addition, AMD-related inflammation andassociated deregulation of complement activity, as indicated byelevation of complement activation products in systemic circulation andin AMD tissues by histopathological analysis, occurs in the absence ofknown genetic polymorphisms in complement proteins. New discoveries,have highlighted the potential pathological impact of complement by theidentification of and presence of the membrane attack complex indiseased tissue and in occurrence of advanced forms of AMD (Whitmore S,et al. 2014, “Complement activation and choriocapillaris loss in earlyAMD: Implications for pathophysiology and therapy.” Progress in Retinaland Eye Research, Dec. 5, 2014 EPub ahead of print; Nishigauchi K M, etal. 2012 “C9-R95X polymorphism in patients with neovascular age-relatedmacular degeneration”, January 131; 53(1) 508-12). These results suggestthe viability of blocking the final complement pathway component as atherapeutic target for treating AMD. To date most therapeutics targetingformation of MAC do so by blocking the formation of C5b the key buildingblock required to initiate MAC formation. However, in doing so they alsoblock formation of C5a resulting in loss of C5a functional activity thathas been associated with tissues homeostasis (removal of opsinizedparticles), neural survival and promotion of an anti-angiogenicresponse. In man, this process of selectively blocking MAC formation isusually carried out by the cell surface protein CD59 which blocks MACassembly and by the soluble factors vitronectin and clusterin. In orderto mimic the natural mechanism and preserve favorable upstreamactivities of complement activation the current application reveals thedevelopment of a novel therapeutic monoclonal antibody that binds C5 butuniquely allows processing of the C5 molecule to C5a and C5b butinhibits formation of MAC, FIG. 2, thus preventing formation of the keypathogenic component associated with AMD. Through blocking MACformation, while preserving key supportive ocular tissues i.e.,choroicapilars and RPE, function and survival of the neural retina,which is vital to maintaining vision will be retained.

SUMMARY OF THE INVENTION

The invention encompasses methods and compositions of a pharmaceuticalformulation comprising an anti-complement C5 antibody or anti-C5antibody. In one aspect, the anti-C5 antibody does not bind to C5a andinhibits complement dependent hemolysis. In another aspect, the anti-C5antibody binds to C5b and inhibits the formation of membrane attackcomplex (MAC) in a patient. In one embodiment, the anti-C5 antibodyblocks C5 binding to human complement component 6. In anotherembodiment, the anti-C5 antibody blocks C5 binding to human complementcomponent 7. In another aspect, the anti-C5 antibody is characterized bythe feature that it no longer binds or has reduced binding to C5 (or asubunit thereof) once it is incorporated into the membrane attackcomplex.

In another aspect, the anti-complement C5 antibody or anti-C5 antibodybinds to C5 with a Kd of less than about 10 pM. In another aspect, theanti-C5 antibody is a monoclonal antibody. In another embodiment, theanti-C5 antibody is selected from the group consisting of a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanizedantibody, a chimeric antibody, a multispecific antibody and an antibodyfragment. In one embodiment, the anti-C5 antibody is an antibodyfragment and that antibody fragment is a Fab fragment, a Fab′ fragment,a F(ab′)2 fragment, a Fv fragment, a diabody, or a single chain antibodymolecule. In another embodiment, the anti-C5 antibody is an IgG1, IgG2,IgG3, or IgG4. In another embodiment, the anti-C5 antibody is an IgG1.

In another aspect, the anti-C5 antibody is coupled to a labelling group.In another embodiment, the anti-C5 antibody is coupled to a labellinggroup and that labelling group is an optical label, radioisotope,radionuclide, an enzymatic group, and a biotinyl group.

In another aspect, the invention comprises a process for preparing anisolated antibody that binds to complement C5 comprising isolating saidantibody from a host cell that secretes the antibody.

In another aspect, the invention is an anti-complement C5 antibodycomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13, 18, 23, 28, 33, and 38. In another aspect, the anti-C5antibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 19, 24, 29, 34 and 39. In another aspect,the anti-C5 antibody comprises an amino acid sequence selected from thegroup consisting of GTS, SGS, RTS, YTS, and WAS. In another aspect, theanti-C5 antibody comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15, 20, 25, 30, 35 and 40. In anotheraspect, the anti-C5 antibody comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 16, 21, 26, 31, 36, and 41. Inanother aspect, the anti-C5 antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 17, 22, 27, 32, 37 and42. In another aspect, the invention is an antibody comprising a firstand second amino acid sequence, the first amino acid sequence comprisinga CDR1 selected from the group consisting of SEQ ID NOs: 13, 18, 23, 28,33, and 38; a CDR2 selected from the group consisting of amino acidsequence GTS, SGS, YTS, and WAS; a CDR3 selected from the groupconsisting of SEQ ID NOs: 14, 19, 24, 29, 34 and 39; and a second aminoacid sequence comprising a CDR1 selected from the group consisting ofSEQ ID NOs: 15, 20, 25, 30, 35 and 40; a CDR2 selected from the groupconsisting of SEQ ID NOs: 16, 21, 26, 31, 36 and 41; and a CDR3 selectedfrom the group consisting of SEQ ID NOs: 17, 22, 27, 32, 37 and 42. Inother embodiment, the invention is an antibody comprising the amino acidsequence of SEQ ID NO: 10 and SEQ ID NO: 2.

In another aspect, the invention comprises a nucleic acid moleculeencoding an isolated antibody that binds to complement C5. In oneembodiment, the nucleic acid molecule encoding the antibody that bindsto complement C5 is operably linked to a control sequence.

In another aspect, the invention comprises an anti-complement C5antibody and a pharmaceutically acceptable carrier. In one embodiment,the anti-complement C5 antibody further comprises an additional activeagent. In another embodiment, the anti-complement C5 antibody andadditional active agent also include a pharmaceutically acceptablecarrier.

In another aspect, the invention comprises a method for treating orpreventing an indication in a patient in need of treatment orprevention, the method comprising administering to the patient, aneffective amount of at least one anti-complement C5 antibody. In oneembodiment, the indication is age-related macular degeneration (AMD). Inanother embodiment, the disease or disorder in a patient in need oftreatment or prevention is an ocular condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the Complement Pathway.

FIG. 2 shows a schematic of MAC formation and shows the mechanism of amonoclonal antibody therapeutic in blocking MAC but not C5a generation

FIG. 3 shows percent inhibition of MAC by anti-C5 antibody sub-clones.

FIGS. 4A and 4B show percent inhibition of MAC by anti-C5 antibodysub-clones.

FIGS. 5A, 5B, and 5C show percent inhibition of MAC by anti-C5 antibodysub-clones.

FIGS. 6A, 6B and 6C show the generation of C5a inhibition by examiningsingle point determinations or by titration of the antibody.

FIG. 7 shows dose dependent interaction of monoclonal antibodies with C5directly coated on to an ELISA plates.

FIG. 8 shows binding affinities of anti-C5 monoclonal antibodies to C5.

FIG. 9 shows the binding of the monoclonal antibodies to C5 protein insolution using Bio-Layer Interferometry (BLI).

FIG. 10 shows ability to recognize C5 with in the C5b-9 complex whendeposited into the bottom of ELISA plates after complement activationwith IgM.

FIGS. 11A and 11B show the ability of the monoclonal antibodies to bindsoluble C5b-9 using Bio-Layer Interferometry (BLI) technology.

FIGS. 12A, 12B and 12C show inhibition of MAC for full-length antibodieswith humanized heavy and light chains of 10C9.

FIGS. 13A, 13B, and 13C show activity of Fab fragments with humanizedheavy and light chains of 10C9.

FIGS. 14 A and 14B shows the H5L2 (humanized 10C9) antibody is effectivein a non-human primate light injury model in blocking complementdeposition in retina (FIG. 14A) and choroid (FIG. 14B) relative tocontrol.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification etc.Enzymatic reactions and purification techniques can be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., entirely incorporated by reference. Unless specificdefinitions are provided, the nomenclature used in connection with, andthe laboratory procedures and techniques of, molecular biology,biological chemistry, physical and bio-physical chemistry, analyticalchemistry, organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques can be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

The following definitions are used herein:

“AMD” refers to all forms of age related macular degeneration inclusiveof but not limited to disease onset, (i.e. early and late), Diseasestage (i.e early, intermediate or advance), Disease type (geographicatrophy or neovascular maculopathy), Disease distribution (ie.Unilateral, Bilateral, Central or Peripheryl), or presence/absence ofdrusen deposits, presence/absence of reticular pseudodrusen, retinalpigment epithelium abnormalities, photoreceptor abnormalities, atrophicage-related macular degeneration, geographic atrophy (GA) andneovascular maculopathy.

“Protein,” as used herein, is meant to refer to at least two covalentlyattached amino acids, and is used interchangeably with polypeptides,oligopeptides, and peptides. The two or more covalently attached aminoacids are attached by a peptide bond.

“C5” refers to human complement Component 5. As used herein, Factor C5,Component Factor 5 are synonymous with C5.

“C5a” refers the smaller fragment of C5 having approximately 77-74 aminoacids and being about 7 kDa, that is produced when C5 is cleaved by C5convertase when activated in the complement cascade. “C5b,” refers tothe larger fragment of C5 that is produced when cleaved by C5 convertasewhen activated in the complement cascade. C5b consists of an alpha chain(about 104 kDa) and a beta chain (about 75 kDa) linked by a singledisulfide residue.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense to refer to a protein, comprising one or morepolypeptide chains that interact with a specific antigen, throughbinding of a plurality of CDRs on the antibody and an epitope of theantigen. An antibody can be a monoclonal (for e.g., full length orintact monoclonal antibodies), polyclonal, multivalent, and/ormultispecific (e.g., bispecific antibodies so long as they exhibit thedesired biological activity). Antibodies can also be or include antibodyfragments (as described herein).

“Epitope” is used to refer to a sequence, structure, or moiety that isrecognized and bound by an antibody. An epitope can be referred to as an“antigenic site.”

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, most or all, of the functionsnormally associated with that portion when present in an intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.In one embodiment, an antibody fragment comprises an antigen bindingsite of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcR binding, antibody half-life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

“Monoclonal” as used herein refers to an antibody obtained from apopulation of cells, wherein the population of cells is clonally-derivedfrom a single parent cell. Monoclonal antibodies are homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical in that they are derived from the same genes and have thesame amino acid sequence and protein structure except for possiblenaturally-occurring mutations that can be present in minor amounts andpost-translational modifications that may, in some cases, be different.Monoclonal antibodies can, in some embodiments, be highly specific. Insome embodiments, a monoclonal antibody can be directed against a singleantigenic site. Furthermore, in contrast to other antibody preparationswhich typically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed againstthe same epitope on the antigen. Individual monoclonal antibodies can beproduced by any particular method. For example, the monoclonalantibodies to be used in accordance with the present disclosure can bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256:495, or can be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), or from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature 352:624-628 andMarks et al. (1991) J. Mol. Biol. 222:581-597.

“Polyclonal” is used to describe a heterogeneous population ofantibodies derived from a heterogeneous population of parent,antibody-producing cells. In most cases the polyclonal antibodies havedifferent affinity for differing epitopes and are produced from geneswith differing sequences.

“Chimeric” antibodies are antibodies comprising amino acid sequencesderived from two or more different species.

“Humanized” antibodies are chimeric antibodies derived from a non-humanparent antibody. In many cases specific amino acid positions in ahumanized antibody, have been changed to correspond to the identity ofthe amino acid at a corresponding position in a human antibody. In manycases, positions in a variable region of the parent (non-human) antibodyare replaced with amino acids from a variable region of a human species.This creates a humanized mouse, rat, rabbit or nonhuman-primate antibodyhaving the desired specificity, affinity, and capacity.

“Variant” refers to sequences that comprise at least one differencecompared to a parent sequence. A variant polypeptide is a protein havingat least about 75% amino acid sequence identity to a parent sequence. Avariant protein can have at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity, or at least about 98% amino acid sequenceidentity, or at least about 99% amino acid sequence identity with aparent amino acid sequence. In some cases variant antibodies areantibodies having one or more difference(s) in amino acid sequence ascompared to a parent antibody. Humanized and chimeric antibodies arevariant antibodies. Variant antibodies, therefore, comprise less than100% sequence identity with a parent antibody. Variant nucleotidesequences comprise less than about 100% sequence identity with a parentnucleotide sequence.

“Isolated” or “purified” refers to a molecule that has been separatedand/or recovered from at least one component of its natural environment,wherein the component is a material that can interfere with the use, oractivity, of the molecule. Components include peptides, sugars, nucleicacids, enzymes, hormones, and other proteinaceous or nonproteinaceoussolutes.

“Complementarity Determining Regions” (CDRs) refers to one or moreregions within an antibody wherein the residues of one or more CDR aidin antigen binding. In many cases, individual amino acids of the CDRscan be in close proximity to atoms of the target antigen. In someembodiments the CDR may be located in an immunoglobulin that may becomprised of three CDR regions. In some cases, as where there are morethan one CDR sequence in a larger amino acid sequence, the CDRs may beseparated by other sequences, and the CDRs numbered. In some cases,multiple CDRs are identified as CDR1, CDR2 and CDR3. Each CDR maycomprise amino acid residues from a Complementarity Determining Regionas defined by Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). Amino acid numbering ofCDRs, as well as other sequences within an antibody, or antibodyfragment is according to that of Kabat. In many cases, CDRs can bedefined by their position in a variable region sequence (numbering as inKabat), for example the light chain CDR 1 may comprise the amino acidsequence between position 24 and position 33; between position 50 andposition 56 for LC CDR2; and between position 89 and position 97 for LCCDR 3; and the heavy chain CDRs may lie between position 26 and position33 for CDR1; position 50 and position 66 for HC CDR 2; and betweenposition 97 and position 103 for HC CDR 3. and/or hypervariable loopsmay lie between light chain residues 26-32 (LC CDR1), residues 50-52 (LCCDR2) and residues 91-96 (LC CDR3); and heavy chain residues 26-32 (HCCDR1), residues 53-55 (HC CDR2) and residues 97-101 (HC CDR3). In someinstances, a Complementarity Determining Region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. In some embodiments, as in where the antibody is a single chainimmunoglobulin, there may be more than one CDR, more than two CDRs, morethan three CDRs, more than four CDRs, or more than five CDRs. In someembodiments, an antibody may be comprised of six CDRs.

“Framework regions,” FRs, are variable domain residues other than theCDR residues. In most embodiments a variable domain has between two andfour FRs identified sequentially. For example a variable regioncomprising three CDRs, has four FRs: FR1, FR2, FR3 and FR4. Where theCDRs are defined according to Kabat, the light chain FR residues arepositioned at about residues 1-23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3),and 98-107 (LCFR4) and the heavy chain FR residues are positioned aboutat residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96 (HCFR3), and 104-113(HCFR4) in the heavy chain residues. If the CDRs comprise amino acidresidues from hypervariable loops, the light chain FR residues arepositioned about at residues 1-23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3),and 98-107 (LCFR4) in the light chain and the heavy chain FR residuesare positioned about at residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96(HCFR3), and 104-113 (HCFR4) in the heavy chain residues. In someinstances, when the CDR comprises amino acids from both a CDR as definedby Kabat and those of a hypervariable loop, the FR residues will beadjusted accordingly. For example, when HC CDR1 includes amino acidsH26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2residues are at positions 36-49.

“Variable domain” refers to portions of a light chain and a heavy chainof traditional antibody molecule that includes amino acid sequences ofComplementarity Determining Regions (CDRs), and Framework Regions (FRs).VH refers to the variable domain of the heavy chain. VL refers to thevariable domain of the light chain.

“Fv” or “Fv fragment” refers to an antibody fragment which contains acomplete antigen recognition and binding site, comprising the FR and CDRsequences. In many embodiments, the Fv consists of a dimer of one heavyand one light chain variable domain in tight association, which can becovalent in nature, for example in a single chain Fv molecule (scFv).The three CDRs of each variable domain interact to define an antigenbinding site on the surface of the VH-VL polypeptide. Collectively, thesix CDRs or a subset thereof confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has, in some cases,the ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

“Fab” or “Fab fragment” contains a variable and constant domain (CL) ofthe light chain and a variable domain and the first constant domain(CH1) of the heavy chain. F(ab′)2 antibody fragments comprise a pair ofFab fragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Percent (%) amino acid sequence homology” is defined as the percentageof amino acid residues in a candidate sequence that are homologous withthe amino acid residues in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence homology. This method takes into account conservativesubstitutions. Conservative substitutions are those substitutions thatallow an amino acid to be substituted with a similar amino acid. Aminoacids can be similar in several characteristics, for example, size,shape, hydrophobicity, hydrophilicity, charge, isoelectric point,polarity, aromaticity, etc. Alignment for purposes of determiningpercent amino acid sequence homology can be achieved in various waysthat are within the ordinary skill of those persons of skill in the art.In some cases, amino acid sequences can be aligned using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. Sequence homology is then calculated relative to the longersequence, i.e. even if a shorter sequence shows 100% sequence identitywith a portion of a longer sequence, the overall sequence identity willbe less than 100%.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Alignment for purposes of determining percentnucleic acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.Sequence identity is then calculated relative to the longer sequence,i.e. even if a shorter sequence shows 100% sequence identity with aportion of a longer sequence, the overall sequence identity will be lessthan 100%.

“Activity” or “biological activity” of a molecule can depend upon thetype of molecule and the availability of tests for assaying a givenactivity. For example, in the context of a C5 antibody, activity refersto its ability to partially or fully inhibit a biological activity ofC5, for example, binding to other complement proteins, cleavage byprotease as exemplified by C5 convertase or other known protease of theextrinsic activation pathway capable of cleaving C5 (Krisinger M. J. etal., Thrombin generates previously unidentified C5 products that supportthe terminal complement activation pathway. Blood, 2012 120(8)1717-1725), or MAC formation. A preferred biological activity of theclaimed C5 antibody is the ability to block processes associated withactivation of the C5 molecule. Preferably the inhibitory activity willachieve a measurable improvement in the state, e.g. pathology, of aC5-associated disease or condition, such as, for example, acomplement-associated eye condition. In some cases, the activityinhibited by the disclosed anti-C5 antibody is through blocking a C5protease or C5 cleavage. In other cases the activity is the ability tobind other complement proteins in a complex preventing membraneinsertion and cell lysis. In some embodiments, the activity of thedisclosed anti-C5 antibody is measured by its ability to inhibithemolysis, C5a generation, MAC formation or association of othercomplement proteins with C5. The activity can be determined through theuse of in vitro or in vivo tests, including binding assays, MACformation assay, generation of complement split products, induction ofcytokine release, or through the use of a relevant animal model, orhuman clinical trials.

“Complement-associated eye condition” is used in the broadest sense andincludes all eye conditions the pathology of which involves complement,activated by either the classical, lectin, alternative or extrinsicpathways. Complement-associated eye conditions include, withoutlimitation, macular degenerative diseases, such as all stages ofage-related macular degeneration (AMD), including dry and exudative(non-exudative and exudative) forms, choroidal neovascularization (CNV),uveitis, diabetic and other ischemia-related retinopathies includingdiabetic macular edema, Central Retinal Vein Occlusion (CRVO), BranchedRetinal Vein Occlusion (BRVO), and other intraocular neovasculardiseases, such as diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, cornealneovascularization, and retinal neovascularization. A preferred group ofcomplement-associated eye conditions includes age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)AMD, choroidal neovascularization (CNV), Macular Telangiectasia,uveitis, diabetic and other ischemia-related neovascular-relatedretinopathies, or cellular degenerative diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Doyne honeycomb retinal dystrophy/Malattia Leventinese, Stargartsdisease, Glucoma, Central Retinal Vein Occlusion (CRVO), BRVO, cornealneovascularization, retinal neovascularization.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatpossesses the desired pharmacological activity of the parent compound.Such salts include acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; andsalts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N methylglucamine, andthe like. In certain embodiments, a pharmaceutically acceptable salt isthe hydrochloride salt. In certain embodiments, a pharmaceuticallyacceptable salt is the sodium salt.

“Pharmaceutically acceptable excipient” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable vehicle, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which a compoundprovided by the present disclosure can be administered to a patient,which does not destroy the pharmacological activity thereof and which isnon-toxic when administered in doses sufficient to provide atherapeutically effective amount of the compound or a pharmacologicallyactive metabolite thereof.

“Treatment” is an administration of at least one therapeutic agent forpreventing the development or altering the pathology of a disorder.Accordingly, treatment refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. As disclosed herein, the preferred agentfor administration comprises at least one of the disclosed anti-C5antibodies. In treatment of a complement related disease, thetherapeutic agent, comprising at least one of the presently disclosedantibodies or a coding sequence for such antibody, may directly orindirectly alter the magnitude of response of a component of thecomplement pathway, or render the disease more susceptible to treatmentby other therapeutic agents, e.g., antibiotics, antifungals,anti-inflammatory agents, chemotherapeutics, etc.

“Therapeutically effective amount” refers to the amount of an agentthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to effect suchtreatment of the disease or symptom thereof. The specifictherapeutically effective amount may vary depending, for example, on theagent, the disease and/or symptoms of the disease, severity of thedisease and/or symptoms of the disease, the age, weight, and/or healthof the patient to be treated, and the judgment of the prescribingphysician. An appropriate amount in any given compound can beascertained by those skilled in the art and/or is capable ofdetermination by routine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease in a patient. A therapeuticallyeffective dose may vary from agent to agent and/or from patient topatient, and may depend upon factors such as the condition of thepatient and the severity of the disease. A therapeutically effectivedose can be determined in accordance with routine pharmacologicalprocedures known to those skilled in the art.

“Pathology” of a disease, such as a complement-associated eye condition,includes all phenomena that compromise the well-being of the patient.This includes, without limitation, abnormal or uncontrollable cellgrowth, protein production, abnormal or uncontrolled cell death,auto-antibody production, complement production, complement activation,MAC formation, interference with the normal functioning of neighboringcells, release of cytokines or other secretory products at abnormallevels, suppression or aggravation of any inflammatory or immunologicalresponse, infiltration of inflammatory cells into cellular spaces, edemaetc.

“Mammal” as used herein refers to any animal classified as a mammal,including, without limitation, humans, higher primates, domestic andfarm animals, and zoo, sports or pet animals such horses, pigs, cattle,dogs, cats and ferrets, etc. In a preferred embodiment of the invention,the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The present disclosure provides antibodies that bind complementComponent 5 protein. Specifically, disclosed herein are antibodies thatbind C5 and C5b, but not C5a. The presently disclosed antibodies do notinhibit C5 cleavage, but do inhibit MAC formation and MAC-dependent celllysis.

The antibodies described herein comprise a scaffold structure with oneor more Complementarity Determining Regions (CDRs). In certainembodiments, the CDRs include no more than two amino acid additions,deletions, or substitutions from one or more of the heavy chain CDR1,CDR2, and CDR3, and the light chain CDR1, CDR2 and CDR3 of a parentsequence, for example SEQ ID NOs:13-48.

In other embodiments, the CDRs are defined by a consensus sequencehaving common conserved amino acid sequences and variable amino acidsequences as described herein.

In certain embodiments, the scaffold structure of the C5 antibodies ofthe disclosure can be based on antibodies, including, but not limitedto, monoclonal antibodies, bispecific antibodies, minibodies, domainantibodies, synthetic antibodies (e.g. antibody mimetics), chimericantibodies, humanized antibodies, antibody fusions (e.g. antibodyconjugates), and fragments of each, respectively. The various structuresare further described and defined hereinbelow. In some embodiments, thescaffold structures comprise one or more of SEQ ID NOs:1-12. In certainembodiments, the scaffold sequences include one or more amino acidadditions, deletions, or substitutions compared to SEQ ID NOs:1-12.

The anti-C5 antibodies are useful in treating consequences, symptoms,and/or the pathology associated with complement activation. Theseinclude, but are not limited to, atherosclerosis, ischemia-reperfusionfollowing acute myocardial infarction, Henoch-Schonlein purpuranephritis, immune complex vasculitis, rheumatoid arthritis, arteritis,aneurysm, stroke, cardiomyopathy, hemorrhagic shock, crush injury,multiple organ failure, hypovolemic shock and intestinal ischemia,transplant rejection, cardiac Surgery, PTCA, spontaneous abortion,neuronal injury, spinal cord injury, myasthenia gravis, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis, GuillainBarre syndrome, Parkinson's disease, Alzheimer's disease, acuterespiratory distress syndrome, asthma, chronic obstructive pulmonarydisease, transfusion-related acute lung injury, acute lung injury,Goodpasture's disease, myocardial infarction, post-cardiopulmonarybypass inflammation, cardiopulmonary bypass, septic shock, transplantrejection, xeno transplantation, burn injury, systemic lupuserythematosus, membranous nephritis, cerebral malaria, Berger's disease,psoriasis, pemphigoid, dermatomyositis, anti-phospholipid syndrome,inflammatory bowel disease, hemodialysis, leukopheresis, plasmapheresis,heparin-induced extracorporeal membrane oxygenation LDL precipitation,extracorporeal membrane oxygenation leukopheresis, plasmapheresis,heparin-induced extracorporeal membrane oxygenation LDL precipitation,extracorporeal membrane oxygenation and the like.

Other uses for the disclosed antibodies include, for example, diagnosisof complement- and C5-associated diseases.

Aspects of the present disclosure provide anti-C5 antibodies,particularly antibodies that include at least one CDR including heavychain and/or light chain CDRs, as more fully described below, orcombinations thereof.

In one aspect, the anti-C5 antibodies inhibit activity of C5 and/or C5b,and inhibit the ability of C5b to form protein complexes. Without beingheld to a particular mechanism or theory, in some embodiments theantibodies interrupt the complement pathway, thereby interrupting thecomplement cascade, formation of the MAC, and cell lysis. Thisdisruption may prevent or alter disease course in, but is not limitedto, geographic atrophy and exudative AMD, uveitis, diabetic and otherneovascular or ischemia-related retinopathies, diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Retinal Angiomatous Proliferation, Central Retinal Vein Occlusion(CRVO), Branched Retinal Vein Occlusion (BRVO), cornealneovascularization, retinal neovascularization, and the like. In someembodiments, the anti-C5 antibody may inhibit C5b initiation of MACformation.

The antibodies of the disclosure thus may serve to identify conditionsrelated to C5 or the complement system or related diseases orconditions. In addition, the antibodies can be used to regulate and/orsuppress effects mediated by C5 and/or other, downstream, complementproteins, as such having efficacy in the treatment and prevention ofvarious diseases and conditions associated with complement and/or C5.

More specifically, the disclosure provides anti-C5 antibodies andpolynucleotides that encode them. In various aspects, the anti-C5antibodies inhibit at least one of the biological responses mediated byC5, C5b and/or other complement proteins, and as such can be useful forameliorating the effects of complement-associated and C5-associateddiseases and disorders. Also provided by the disclosure are expressionsystems, including mammalian cell lines and bacterial cells, for theproduction of anti-C5 antibodies and methods of treating diseasesassociated with complement activation.

The antibodies of the present disclosure comprise a scaffold structureand one or more complementary determining regions (CDRs) that bind toC5. In various embodiments, the antibody comprises a first and/or secondamino acid sequence.

In one embodiment, the first and/or the second amino acid sequencecomprises a sequence selected from the group consisting of SEQ IDNOs:1-48.

In various embodiments, the antibodies can include one or both of thefirst and second amino acid sequences. The first and second amino acidsequences can be a single linear amino acid sequence, can be covalentlybonded by disulfide bridges, or can be non-covalently bonded.

Complement Component 5, C5

The membrane attack complex (MAC) is typically formed as a result of theactivation of one or more of the three principal pathways, eg thealternative pathway, Lectin pathway or classical pathway of thecomplement system or through alterations in C5 confirmation oractivation by the less common extrinsic pathway. MAC is one of theeffector proteins of the immune system and forms transmembrane channels.These channels disrupt the phospholipid bilayer of target cells, leadingto cell lysis and death. A critical protein in the assembly of the MACis C5. C5 has a molecular weight of about 190 kDa (about 1600 aa) andconsists of two polypeptide chains, the alpha chain (a, 115 kDa) and thebeta chain (β, 75 kDa). The alpha and beta chains are connected bydisulfide bonds. C5 convertase cleaves C5 at an arginine, 75 residuesdownstream from the N-terminus of the alpha-chain. This cleavagereleases the small C5a fragment (approximately 77-74 aa in length andabout 11 kDa), which is a potent inflammatory molecule. The C5convertase cleavage also results in activation of C5b, which can theninitiate formation of the membrane attack complex (MAC). The C5b proteinconsists of the alpha chain (now 104 kDa) and the beta chain (75 kDa).

Cleavage of C5 by the C5 convertase leads to the formation of C5a andC5b. The newly formed C5b fragment recruits C6, followed by thesequential addition of C7, C8 and multiple C9 molecules to assemble MAC.Active MAC has a subunit composition of C5b-C6-C7-C8-C9{n}. The ringstructure formed by C9 is a pore in the membrane of the target cell. Ifenough pores form, the cell is no longer able to survive due to freediffusion of molecules in and out of the cell. At sublyticconcentrations these pores can contribute to proinflammatory cellactivation, while at lytic concentrations pore formation leads to celldeath. The formation of MAC is schematically shown in FIG. 2. Both C5aand C5b are proinflammatory molecules. C5a binds the C5a receptor (C5aR)and stimulates the synthesis and release from human leukocytes ofproinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6 and IL-8.C5a has also been shown to be associated with tissue homeostasis(removal of opsinized particles), neural survival and promotion ofanti-angiogenic response. Most anti-C5 antibodies inhibits the formationof C5a and C5b, which would not only interfere with the activation ofMAC by blocking C5b formation, but would also detrimentally block C5aactivity, which may contribute to maintenance of retinal health. What isneeded is an antibody that selectively blocks C5b so it inhibits MACformation, while preserving the actions of C5a.

Reducing the formation of C5b may aid in treating many diseases of thecomplement system as well as inflammatory diseases. One such disease isage-related macular degeneration or AMD. AMD is a medical condition thatresults in a loss of vision, due to deterioration of the retina. Thecomplement system has been implicated in AMD through a strongassociation between several genes in the complement system and aperson's risk of developing AMD. Thus, inhibiting the complement systemthrough prevention of C5b protein incorporation in the MAC may becritical to the therapeutic treatment of AMD.

Anti-C5 Antibodies

In one aspect, the disclosure provides antibodies that bind C5, do notbind C5a, and do not inhibit the formation of C5a. In certain aspects,the disclosure provides recombinant antibodies that bind C5, i.e.anti-C5 antibodies. In this context, recombinant antibodies can beproduced using recombinant techniques, i.e., through the expression of arecombinant nucleic acid as described below. Methods and techniques forthe production of recombinant proteins are well known in the art.

In some embodiments, the antibodies of the disclosure are isolated orpurified. An isolated or purified antibody can be unaccompanied by atleast some of the material with which it is normally associated in itsnatural state (contaminating material). In a one embodiment, thecontaminating material constitutes less than about 50%, alternativelyless than about 20%, and alternatively less than about 10% by weight ofthe total weight of a given sample. In some embodiments the contaminantmay be protein.

In many embodiments, the purified anti-C5 antibody is produced in orfrom an organism other than the organism from which it is derived. Insome embodiments, the anti-C5 antibody can be made at a significantlyhigher concentration than is normally seen, through the use of aninducible promoter or high expression promoter, such that the antibodyis made at increased concentration levels.

In some embodiments, the isolated or purified antibody can be removedfrom components that can interfere with diagnostic and/or therapeuticuses for the antibody. In some embodiments, the antibody will bepurified to greater than 90% by weight of antibody, wherein the totalprotein concentration is determined, for example by the Lowry method,and the percent antibody concentration is determined by a visual method,such as a protein gel. In one embodiment the anti-C5 antibody is morethan 99% by weight, for example pure enough to obtain at least 15residues of N-terminal or internal amino acid sequence by use of acommon amino acid sequencing technique (e.g. Edman degradation and massspectrometry), or to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedantibodies include antibodies in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

The disclosed antibody can bind specifically to C5 and can be used toinhibit or modulate the biological activity of C5 and C5b. In certainembodiments, the disclosed antibodies are created by immunization of ananimal, in other cases antibodies can be produced by recombinant DNAtechniques. In additional embodiments, anti-C5 antibodies can beproduced by enzymatic or chemical cleavage of traditional antibodies(traditional antibodies may be synonymous with human antibodies). Insome embodiments, the antibody can comprise a tetramer. In some of theseembodiments, each tetramer is typically composed of two identical pairsof polypeptide chains, each pair having one light chain (typicallyhaving a molecular weight of about 25 kDa) and one heavy chain(typically having a molecular weight of about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids and can be responsible for antigenrecognition. The carboxy-terminal portion of each chain can define aconstant region, which is primarily responsible for effector function.Human light chains are classified as kappa and lambda light chains.Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. IgG has several subclasses, including, but not limited toIgG1, IgG2, IgG3, and IgG4.

Some antibodies, for example antibodies found in camels and llamas, canbe dimers consisting of two heavy chains and include no light chains.Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter et al.,2001, J. Biol. Chem. 276:26285-26290. Crystallographic studies of camelantibodies have revealed that the CDR3 regions of these antibodies forma surface that interacts with the antigen and thus is critical forantigen binding like in the more typical tetrameric antibodies. Thedisclosure encompasses dimeric antibodies consisting of two heavychains, or fragments thereof that can bind to and/or inhibit thebiological activity of C5 and/or C5b.

The antibodies of the disclosure specifically bind to human C5. Anantibody can specifically bind to C5 when the antibody has a higherbinding affinity for that C5 than for any other antigen or protein. Invarious embodiments, the binding affinity is measured by determining anequilibrium binding constant, for example a K_(d) (or Kd), or K_(a) (orKa). In some embodiments the disclosed antibody binds to a targetantigen with a Kd from about 10⁻⁷ M to about 10⁻¹³ M, or from about10⁻⁹M to about 10⁻¹² M, or from about 10⁻¹¹ M to about 10⁻¹² M. Invarious embodiments, the Kd is less than about 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M or 10⁻¹²M, and more than about 10⁻¹³ M, 10⁻¹² M, 10⁻¹¹ M, 10⁻¹⁰M, 10⁻⁹ M.

In some cases the Kd for the other antigen is greater than 1× the targetantigen Kd, greater than 2× the target antigen Kd, greater than 3× thetarget antigen Kd, greater than 4× the target antigen Kd, greater than5× the target antigen Kd, greater than 6× the target antigen Kd, greaterthan 7× the target antigen Kd, greater than 8× the target antigen Kd,greater than 9× the target antigen Kd, greater than 10× the targetantigen Kd (for example where the Kd of the antibody is X⁻⁰⁹M for thetarget antigen, the Kd of the antibody for another antigen can be 10×greater, or X⁻⁰⁸ M), or greater than 100× (for example where the Kd ofthe antibody is X⁻¹⁰ M for the target antigen, the Kd of the antibodyfor another antigen can be 10× greater, or X⁻⁰⁸ M).

In some cases, the equilibrium binding constant can be expressed as anequilibrium association constant, K_(a) or Ka.

The equilibrium binding constant can be determined using variousmethods. In some cases, an equilibrium binding constant for thedisclosed antibody is determined by measuring on (k₁) and off (k⁻¹)rates in a protein binding assay. One exemplary method of determiningthe equilibrium binding constant is by Bio-Layer Interferometry (BLI).BLI is a label-free technology capable of determining binding kineticsin solution. In one exemplary method, an antibody can be a human IgG,and the anti-C5 antibody can be captured by an Anti-human IgG Fc capture(AHC) biosensor tips (FortéBio, Menlo Park, Calif., USA) according tothe manufacturers directions. Other types of protein binding assaysinclude: Co-immunoprecipitation; Bimolecular fluorescencecomplementation; Affinity electrophoresis; Pull-down assays; Labeltransfer; The yeast two-hybrid screen; Phage display; in vivocrosslinking of protein complexes using photo-reactive amino acidanalogs; Tandem affinity purification; Chemical cross-linking; Chemicalcross-linking followed by high mass MALDI mass spectrometry; SPINE(Strepprotein interaction experiment); Quantitative immunoprecipitationcombined with knock-down; Proximity ligation assay Bio-LayerInterferometry; Dual polarisation interferometry; Static lightscattering; Dynamic light scattering; Surface plasmon resonance;Fluorescence polarization/anisotropy; fluorescence correlationspectroscopy; Fluorescence resonance energy transfer; Protein activitydetermination by NMR multi-nuclear relaxation measurements, or 2D-FT NMRspectroscopy in solutions, combined with nonlinear regression analysisof NMR relaxation or 2D-FT spectroscopy data sets; Protein—proteindocking; Isothermal Titration calorimetry; and, MicroscaleThermophoresis.

In embodiments where the anti-C5 antibody is used for therapeuticapplications, one characteristic of an anti-C5 antibody is that it canmodulate and/or inhibit one or more biological activities of, ormediated by C5. In this case, the antibody can bind specifically to C5,can substantially modulate the activity of C5 and/or C5b, and/or caninhibit the binding of C5b to other proteins (e.g. C6, C7).

In many embodiments, C5 activity, and the antibody's ability to inhibitthat activity, is measured by analyzing lysis of red blood cells in thepresence of 10% human serum. Activation of the alternative pathway of(AP) requires higher concentrations of serum than the classical pathway.Generally, a final concentration of 5 mM Mg′ in the presence of 5 mMEGTA is used in the assays where the EGTA chelates Ca⁺⁺ preferentially.The AP of most mammalian species is activated spontaneously by rabbiterythrocytes so they are a convenient target. Prepare rabbiterythrocytes (Complement Technology, Inc.) by washing 3 times with GVB⁰(CompTech product) and re-suspending into 5×10⁸/ml. Different amount ofanti-factor C5 antibody was diluted with GVB⁰. Mix the 100 ul reactionon ice in the order of serial diluted anti-factor Bb antibody, 0.1MMgEGTA (CompTech product), ½NHS (normal human serum diluted ½ withGVB⁰), and rabbit Er. Then, incubate the reaction at 37° C. for 30minutes on a shaker. Add 1.0 ml cold GVBE. Mix and centrifuge for 3 minat approx. 1000×g, or higher, to pellet cells. Transfer 100 ul of thesupernatant to a 96-well plate and read at 412 nm (SoftMax Pro 4.7.1).Data was analyzed using GraphPad Prism 4.

Not every antibody that specifically binds to an antigen can blockantigen binding to its normal ligand and thus inhibit or modulate thebiological effects of the antigen. As is known in the art, such aneffect can depend on what portion of the antigen the antibody binds, andon both the absolute and the relative concentrations of the antigen andthe antibody, in this case, a C5 antibody. To be considered capable ofinhibiting or modulating the biological activity of C5 and/or C5b, asmeant herein, an antibody can be able, for example, to inhibit the humanserum mediated hemolysis by at least about 20%, 40%, 60%, 80%, 85%, 90%,95%, 99%, or more.

The concentration of an antibody required to inhibit C5 and/or C5bactivity can vary widely and may depend upon how tightly the antibodybinds to C5 and/or C5b. For example, one molecule or less of an antibodyper molecule of C5 can be sufficient to inhibit biological activity. Insome embodiments, a ratio of C5:anti-C5 antibody of about 1,000:1 toabout 1:1,000, including about 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20,1:40, 1:60, 1:100, 1:500, 1:1,000 or more can be required to inhibit thebiological activity of C5. In many cases, the ability to inhibit C5activity may depend upon the concentration of C5 and/or theconcentration of anti-C5 antibody.

In some embodiments, the antibodies of the disclosure comprise (a) ascaffold, and (b) one or more CDRs, which are regions that aredeterminative of antigen binding specificity and affinity. ComplementaryDetermining Regions or CDRs are regions of an antibody that constitutesthe major surface contact points for antigen binding. One or more CDRsare embedded in the scaffold structure of the antibody. The scaffoldstructure of the antibodies of the disclosure can be an antibody, orfragment or variant thereof, or can be completely synthetic in nature.The various scaffold structures of the antibodies of the disclosure arefurther described below.

In an embodiment of the presently disclosed antibodies, the antibody canbe a variant antibody having an amino acid sequence with at least 75%amino acid sequence identity, homology, or similarity with the aminoacid sequence of a parent amino acid sequence. For example, in someembodiments the heavy or light chain variable domain sequence of thevariant antibody is 75% identical to the heavy or light chain variabledomain sequence of a parent sequence, alternatively at least 80%,alternatively at least 85%, alternatively at least 90%, andalternatively at least 95%. In most cases, the variant antibody willhave few or no changes in the CDR sequence, and therefore, in mostcases, will bind the target antigen with a similar affinity. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the variant sequence that areidentical (i.e. same residue) or similar (i.e. amino acid residue fromthe same group based on common side-chain properties, see below) withthe parent antibody amino acid sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity.

CDRs

The antibodies of the disclosure include scaffold regions and one ormore CDRs. An antibody of the disclosure may have between one and sixCDRs (as typically do naturally occurring antibodies), for example, oneheavy chain CDR1 (“HC CDR1” or “CDRH1”), and/or one heavy chain CDR2(“HC CDR2” or “CDRH2”), and/or one heavy chain CDR3 (“HC CDR3” or“CDRH3”), and/or one light chain CDR1 (“LC CDR1” or “CDRL1”), and/or onelight chain CDR2 (“LC CDR2” or “CDRL2”), and/or one light chain CDR3(“LC CDR3” or “CDRL3”). The term “naturally occurring” as usedthroughout the specification in connection with biological materialssuch as polypeptides, nucleic acids, host cells, and the like, refers tomaterials which are found in nature. In naturally occurring antibodies,a heavy chain CDR1 typically comprises about five (5) to about seven (7)amino acids, a heavy chain CDR2 typically comprises about sixteen (16)to about nineteen (19) amino acids, and a heavy chain CDR3 typicallycomprises about three (3) to about twenty five (25) amino acids. CDR1 ofthe light chain typically comprises about ten (10) to about seventeen(17) amino acids, the light chain CDR2 typically comprises about seven(7) amino acids, and the light chain CDR3 typically comprises aboutseven (7) to about ten (10) amino acids.

Amino acids of the present disclosure include natural and syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine). Such synthetic amino acids can be incorporated, inparticular when the antibody is synthesized in vitro by conventionalmethods well known in the art. In addition, any combination ofpeptidomimetic, synthetic and naturally occurring residues/structurescan be used. Amino acid includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” can be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration. In some embodiments,the amino acids can form peptidomimetic structures, i.e., peptide orprotein analogs, such as peptoids (see, Simon et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:9367, incorporated by reference herein), which canbe resistant to proteases or other physiological and/or storageconditions.

The structure and properties of CDRs within a naturally occurringantibody are described further below. Briefly, in a traditional antibodyscaffold, the CDRs are embedded within a framework in the heavy andlight chain variable region where they constitute the regionsresponsible for antigen binding and recognition. A variable regioncomprises at least three heavy or light chain CDRs, see, supra (Kabat etal., 1991, Sequences of Proteins of Immunological Interest, PublicHealth Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987,J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883),within a framework region (designated framework regions 1-4, FR1, FR2,FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk,1987, supra). See, infra. The CDRs provided by the present disclosure,however, may not only be used to define the antigen binding domain of atraditional antibody structure, but can be embedded in a variety ofother scaffold structures, as described herein.

Specific CDRs for use in the disclosed antibodies are presented in Table1.

TABLE 1 CDR Amino Acid Sequence for AntibodiesLIGHT CHAIN VARIABLE DOMAIN CDRs 1B6 VL CDR1 SEQ ID NO: 13 SSISSSNVL CDR2 SEQ ID NO: 14 GTS VL CDR3 SEQ ID NO: 15 QQWSSYPFT 6C12 VL CDR1SEQ ID NO: 19 SSISSSN VL CDR2 SEQ ID NO: 20 GTS VL CDR3 SEQ ID NO: 21QQWSTYPFT 8c7 VL CDR1 SEQ ID NO: 25 KSISKY VL CDR2 SEQ ID NO: 26 SGSVL CDR3 SEQ ID NO: 27 QQHNEYPYT 10B11 VL CDR1 SEQ ID NO: 31 SSISSNYVL CDR2 SEQ ID NO: 32 RTS VL CDR3 SEQ ID NO: 33 QQGSGIFT 10G4 VL CDR1SEQ ID NO: 37 QDISSY VL CDR2 SEQ ID NO: 38 YTS VL CDR3 SEQ ID NO: 39QQGNVFPWT 10C9 VL CDR1 SEQ ID NO: 43 QDVNTA VL CDR2 SEQ ID NO: 44 WASVL CDR3 SEQ ID NO: 45 QQHHVSPWT HEAVY CHAIN VARIABLE DOMAIN CDRs 1B6VH CDR1 SEQ ID NO: 16 GYTFTDYE VH CDR2 SEQ ID NO: 17 IDPETGGA VH CDR3SEQ ID NO: 18 TRLGSSPWYFDV 6C12 VH CDR1 SEQ ID NO: 22 GYTFTDYE VH CDR2SEQ ID NO: 23 IDPETGGT VH CDR3 SEQ ID NO: 24 TRLGISPWYFDV 8c7 VH CDR1SEQ ID NO: 28 GYRFTDYN VH CDR2 SEQ ID NO: 29 ISPNNGGT VH CDR3SEQ ID NO: 30 ARREAWYGGYYKWYFDV 10B11 VH CDR1 SEQ ID NO: 34 GYTFTTYGVH CDR2 SEQ ID NO: 35 INTYSGVP VH CDR3 SEQ ID NO: 36 ARRDFYGNYGDY 10G4VH CDR1 SEQ ID NO: 40 GYTFTDSY VH CDR2 SEQ ID NO: 41 ILPNNGGI VH CDR3SEQ ID NO: 42 ARSGGLVGGYFDY 10C9 VH CDR1 SEQ ID NO: 46 GYTFTDEY VH CDR2SEQ ID NO: 47 INPNNGGA VH CDR3 SEQ ID NO: 48 ARLGYSNPYFDF

In another embodiment, the disclosure provides an antibody that bindsC5, wherein said antibody comprises at least one HC CDR region having nomore than two (2) amino acid additions, deletions or substitutions ofany of SEQ ID NOs:16-18, 22-24, 28-30, 34-36, 40-42, and 46-48 and/or atleast one LC CDR region having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:13-15, 19-21,25-27, 31-33, 37-39, and 43-45. Embodiments of various heavy chain andlight chain variable regions of the disclosure are depicted in TABLE 2and SEQ ID NOs:1-12. In some embodiments, of particular use areantibodies with a HC CDR3 and/or LC CDR3 region. Additionally, in someembodiments antibodies can have one CDR having no more than two (2)amino acid additions, deletions or substitutions of the sequenceselected from the HC CDR regions of any of SEQ ID NOs:16-18, 22-24,28-30, 34-36, 40-42, and 46-48 and a LC CDR having no more than two (2)amino acid additions, deletions, or substitutions of any of SEQ IDNOs:13-15, 19-21, 25-27, 31-33, 37-39, and 43-45 (e.g., the antibody hastwo CDR regions, one HC CDR and one LC CDR, a specific embodiment areantibodies with both a HC CDR3 and a LC CDR3, for example, SEQ ID NOs:45and 48).

TABLE 2 Light Chain Sequences SEQ ID NO: 1, L1DIVLTQSPDSLAVSLGERATINCKASQDVNTAVAWYQQKPDQSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQAEDVAVYFCQQHHVSPWTFGG GTKVEIKSEQ ID NO: 3, L2 DIVLTQSPATLSLSPGERATLSCRASQDVNTAVAWYQQKPDQSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQPEDFAVYFCQQHHVSPWTFGG GTKVEIKSEQ ID NO: 5, L3 DIVLTQSPSFLSASVGDRVTITCQASQDVNTAVAWYLQKPGKSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQPEDFAVYFCQQHHVSPWTFGG GTKVEIKSEQ ID NO: 7, L4 DIVLTQSPATLSLSPGERATLSCRASQDVNTAVAWYQQKPGKSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQPEDFAVYFCQQHHVSPWTFGG GTKVEIKSEQ ID NO: 9, L5 DIVLTQSPATLSLSPGERATLSCRASQDVNTAVAWYQQKPGQSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQSEDFAVYFCQQHHVSPWTFGG GTKVEIKSEQ ID NO: 11, L6 DIVLTQSPSFLSASVGDRVTITCQASQDVNTAVAWYQQKPGKSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQPEDFAVYFCQQHHVSPWTFGG GTKVEIKHeavy Chain Sequences SEQ ID NO: 2, H1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDEYMNWVRQAPGQSLEWMGYINPNNGGADYNQKFQGRVTMTVDQSISTAYMELSRLRSDDTAVYFCARLG YSNPYFDFWGQGTLVTVSSSEQ ID NO: 4, H2 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDEYMNWVRQAPGKSLEWVGYINPNNGGADYNQKFQGRVTITVDQSASTAYMELSSLRSEDTAVYFCARLG YSNPYFDFWGQGTLVTVSSSEQ ID NO: 6, H3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDEYMNWVRQAPGQSLEWMGYINPNNGGADYNPSLKSRVTISVDQSISTAYMELSRLRSDDTAVYFCARLG YSNPYFDFWGQGTLVTVSSSEQ ID NO: 8, H4 EVQLVESGGGLVKPGGSLRLSCAASGYTFTDEYMNWVHQAPGKSLEWVGYINPNNGGADYNPSLKSRVTISVDQSKSIAYLQMNSLKTEDTAVYFCARLG YSNPYFDFWGQGTLVTVSSSEQ ID NO: 10, H5 QVQLKQSGAEVKKPGASVKVSCKASGYTFTDEYMNWVRQAPGKSLEWMGYINPNNGGADYNQKFQGRVTMTVDQSISTAYMELSRLRSDDTAVYFCARLG YSNPYFDFWGQGTLVTVSSSEQ ID NO: 12, H6 QVQLVQSGSELKKPGASVKVSCKASGYTFTDEYMNWVRQAPGKSLEWMGYINPNNGGADYNQKFQGRVTMTVNQSISTAYMELSRLRSDDTAVYFCARLG YSNPYFDFWGQGTLVTVSS

Variant CDR Sequences

In another embodiment, the disclosure provides an antibody that binds aC5 protein, wherein said antibody comprises at least one HC CDR regionhaving no more than two (2) amino acid additions, deletions orsubstitutions of any HC CDR1, HC CDR2, or HC CDR3 region (as discussedabove) of SEQ ID NOs:16-18, 22-24, 28-30, 34-36, 40-42, and 46-48 and/orat least one LC CDR region having no more than two (2) amino acidadditions, deletions or substitutions of any LC CDR1, LC CDR2, or LCCDR3 region (as discussed above) of SEQ ID NOs:13-15, 19-21, 25-27,31-33, 37-39, and 43-45. In this embodiment, of particular use areantibodies with a HC CDR3 or LC CDR3 region. Additional embodimentsutilize antibodies with one CDR having no more than 2 amino acidadditions, deletions or substitutions of the sequence selected from theHC CDR regions of any of SEQ ID NOs:16-18, 22-24, 28-30, 34-36, 40-42,and 46-48 and a LC CDR region having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:13-15, 19-21,25-27, 31-33, 37-39, and 43-45 (e.g., the antibody has two CDR regions,one HC CDR and one LC CDR, a specific embodiment are antibodies withboth a HC CDR3 and a LC CDR3 region, for example SEQ ID NO:45 and 48).

As will be appreciated by those in the art, for any antibody with morethan one CDR from the depicted sequences, any combination of CDRsindependently selected from the depicted sequences is useful. Thus,antibodies with one, two, three, four, five or six independentlyselected CDRs can be generated. However, as will be appreciated by thosein the art, specific embodiments generally utilize combinations of CDRsthat are non-repetitive, e.g., antibodies are generally not made withtwo HC CDR2 regions, etc.

A further aspect of the disclosure provides for an isolated antibodythat binds C5 where the isolated antibody comprises a heavy chain aminoacid sequence having no more than two (2) amino acid additions,deletions or substitutions of any of SEQ ID NOs:16-18, 22-24, 28-30,34-36, 40-42, and 46-48, and a light chain amino acid sequence having nomore than two (2) amino acid additions, deletions or substitutions ofany of SEQ ID NOs:13-15, 19-21, 25-27, 31-33, 37-39, and 43-45. It isnoted that any of the heavy chain sequences can be mixed and matchedwith any of the light chain sequences.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs, described herein, is at least 80% when comparedto the sequences disclosed herein. In many cases the aa homology,similarity, or identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, and 99%.

Sequence Identity/Homology

As is known in the art, a number of different programs can be used toidentify the degree of sequence identity or similarity a protein ornucleic acid has to a second sequence.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, using the default settings, or byinspection. Percent identity can be calculated by FastDB based upon thefollowing parameters: mismatch penalty of 1; gap penalty of 1; gap sizepenalty of 0.33; and joining penalty of 30, “Current Methods in SequenceComparison and Analysis,” Macromolecule Sequencing and Synthesis,Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values for proteins:overlap span=1, overlap fraction=0.125, word threshold, T=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values can be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs or variable regions are at least 80% to thesequences, or alternatively increasing homologies or identities of atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost100%.

In a similar manner, percent (%) nucleic acid sequence identity, withrespect to the nucleic acid sequences that encode the disclosedantibodies, is the percentage of nucleotide residues in a candidatesequence that are identical with the nucleotide residues in the codingsequence of the antibody. A specific method utilizes the BLASTN moduleof WU-BLAST-2 set to the default parameters, with overlap span andoverlap fraction set to 1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andvariant variable domain sequences are at least 80%, and alternativelywith increasing homologies or identities of at least 85%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%. In many casesnon-identical nucleic acid sequences, because of the degeneracy of thegenetic code, can code for the same amino acid sequence.

Homology between nucleotide sequences is often defined by their abilityto hybridize to each other. In some embodiments, selective hybridizationcan refer to binding with high specificity. Polynucleotides,oligonucleotides and fragments thereof in accordance with the disclosureselectively hybridize to nucleic acid strands under hybridization andwash conditions that minimize appreciable amounts of detectable bindingto nonspecific nucleic acids. High stringency conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein.

The stringency of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, probe concentration/composition, targetconcentration/composition, washing temperature, and salt concentration.In general, longer probes require higher temperatures for properannealing, while shorter probes need lower temperatures. Hybridizationgenerally depends on the ability of denatured DNA to re-anneal whencomplementary strands are present in an environment below their meltingtemperature. The higher the degree of desired homology between the probeand hybridizable sequence, the higher the relative temperature that canbe used. As a result, it follows that higher relative temperatures wouldtend to make the reaction conditions more stringent, while lowertemperatures less so. For additional details and explanation ofstringency of hybridization reactions, see Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, (1995).

High stringency conditions are known in the art; see, for exampleSambrook et al., 2001, supra, and Short Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, both ofwhich are hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques In Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993).

In some embodiments, stringent or high stringency conditions can beidentified by those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42 C; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium (as the target sequences are present in excess,at Tm, 50% of the probes are occupied at equilibrium). Stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium Ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions can be used, as areknown in the art; see, Sambrook et al., 2001, supra; Ausubel et al.,1992, supra, and Tijssen, 1993, supra.

In some cases, moderately stringent conditions can include the use ofwashing solution and hybridization conditions (e.g., temperature, ionicstrength and % SDS) less stringent that those described above. Anexample of moderately stringent conditions is overnight incubation at37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

In some embodiments, the disclosed antibodies and variants thereof canbe prepared by site specific mutagenesis of nucleotides within a DNAsequence encoding the antibody. This can be achieved using cassette orPCR mutagenesis or other techniques well known in the art, to produceDNA encoding the variant, and thereafter expressing the recombinant DNAin cell culture as outlined herein. In some cases, antibody fragmentscomprising variant CDRs having up to about 100-150 residues can beprepared by in vitro synthesis using established techniques. Thesevariant fragments can exhibit the same qualitative biological activityas the naturally occurring analogue, e.g., binding to C5 and inhibitingcomplement, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize a mutation at a givensite, random mutagenesis can be conducted at the target codon or regionand the expressed antibody CDR or variable region sequence variantsscreened for the optimal desired antibody activity. Techniques formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, M13 primer mutagenesis andPCR mutagenesis. Screening of the mutants is done using assays ofantibody activities, such as C5 binding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions can betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions can be much larger.

Substitutions, deletions, insertions or any combination thereof can beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of theantibody. However, larger changes can be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as Table 3.

TABLE 3 Exemplary Original Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu Ala Ser

Changes in function or immunological identity can be made by selectingsubstitutions that are less conservative than those shown in Table 3.For example, substitutions can be made which more significantly affect:the structure of the polypeptide backbone in the area of the alteration,for example the alpha-helical or beta-sheet structure; the charge orhydrophobicity of the molecule at the target site; or the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the polypeptide's properties are those in which(a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the disclosed C5 antibody, as needed. Alternatively,a variant can be selected wherein the biological activity of thedisclosed antibody is altered. For example, glycosylation sites can bealtered or removed as discussed herein.

Disclosed herein are polypeptide sequences homologous to SEQ IDNOs:1-48. Polypeptides disclosed herein can include amino acid sequencesthat are identical to the disclosed amino acid sequences. In othercases, the claimed polypeptides include amino acid sequences that cancomprise conservative amino acid substitutions as compared to thedisclosed sequence. Conservative amino acid substitutions can includeamino acids that share characteristics with the substituted amino acid.In various cases, conservative substitution can be made withoutsignificant change in the structure or function of the polypeptide.

Conservative amino acid substitutions can be made on the basis ofrelative similarity of side-chain, size, charge, hydrophobicity,hydrophilicity, isoelectric point, etc. In various cases, substitutionscan be assayed for their effect on the function of the protein byroutine testing. Conserved amino acid substitutions include amino acidswith similar hydrophilicity value, as wherein amino acids have ahydropathic index which can be based upon an amino acid's hydrophobicityand charge. In various cases, conserved amino acid substitutions can bemade between amino acids of the same class, for example non-polar aminoacids, acidic amino acids, basic amino acids, and neutral amino acids.Conservative substitutions can also be based upon size or volume. Aminoacids can also be classified based upon their ability to form or break agiven structure, such as an alpha helix, beta sheet, or intra- orinter-molecular interaction. In various cases conservative amino acidsubstitutions are based upon more than one characteristic.

Currently disclosed polypeptides can include both natural andnon-natural amino acids. In various cases, natural amino acid sidechains can be substituted with non-natural side chains. In variouscases, amino acids can be derivatised.

The disclosed polypeptides include polypeptides that are homologous tothe sequences of SEQ ID NOs:1-48. Homology can be expressed as %identity or % similarity or % positive. In various cases, % identity isa percentage of amino acids that are identical between two alignedpolypeptides, and % similar or % positive is a percentage of amino acidsthat are non-identical but represent conservative substitutions. Aconservative substitution may be a substitution of a like-charged aminoacid, a like-sized amino acid, a like-polarity amino acid, etc. Forexample, lysine to arginine can be considered a conservativesubstitution where charge is considered.

In various cases, two polypeptides can be aligned by algorithms, forexample BLASTp. In various cases, the BLASTp parameters can be set witha maximum target sequence length equal to, greater, or less than thelength of the longer of the two polypeptides, the expect threshold canbe set to 10, the word size to 3, and scoring matrix can be BLOSUM62,with gap costs of 11 for existence and 1 for extension. BLASTp canreport homology of aligned polypeptides as “Identities” and “Positives.”The aligned sequences can include gaps to achieve the alignment.

In various cases, homology of amino acid sequences can reflect thepercentage of identity or positives when optimally aligned as describedabove. In various cases, the % homology (% positive) or % identity canbe calculated by dividing the number of aligned amino acids within acomparison window. A comparison window can be the entire length of oneor the other polypeptides, if the two polypeptides are of unequallength. In other cases, the comparison window can be a portion of one ofthe polypeptides. In various cases the comparison window for measuringhomology or identity of two polypeptide sequences is greater than about40 aa (amino acids), 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70 aa, 75 aa, 80aa, 85 aa, 90 aa, 95 aa, 100 aa, 150 aa, or 200 aa, and/or less thanabout 200 aa, 150 aa, 100 aa, 95 aa, 90 aa, 85 aa, 80 aa, 75 aa, 70 aa,65 aa, 60 aa, 55 aa, 50 aa, or 45 aa. In some embodiment, as in the casewith CDR sequences, the comparison window may be less than 40 aa, forexample between less than about 25 aa, 24 aa, 23 aa, 22 aa, 21 aa, 20aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10aa, 9 aa, 8 aa, 7 aa, 6 aa, 5 aa, or 4 aa, and greater than about 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, or 24 aa.

In various cases, the claimed amino acid sequences can have % identityor % homology (% positive) over a given comparison window, that isgreater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% and/or less than about 100%, 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70%.

In various cases, a sequence alignment can be performed using variousalgorithms, including dynamic, local, and global alignment. For example,the algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482; thealignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443;the similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. USA 85: 2444. In various cases, computer programs can implementthese algorithms (such as EMBOSS, GAP, BESTFIT, FASTA, TFASTA BLAST,BLOSUM, etc.).

In alternative cases, conserved amino acid substitutions can be madewhere an amino acid residue is substituted for another in the sameclass, for example where the amino acids are divided into non-polar,acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu,Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His;neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

In some cases, conserved amino acid substitutions can be made where anamino acid residue is substituted for another having a similarhydrophilicity value (e.g., within a value of plus or minus 2.0), wherethe following can be an amino acid having a hydropathic index of about−1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned to amino acidresidues: Arg (+3; 0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3);Asn (+0.2); Gin (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His(−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr(−2.3); Phe (−2.5); and Trp (−3.4).

In alternative cases, conserved amino acid substitutions can be madewhere an amino acid residue is substituted for another having a similarhydropathic index (e.g., within a value of plus or minus 2.0). In suchcases, each amino acid residue can be assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics, as follows:lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9);Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3);Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5);Lys (−3.9); and Arg (−4.5).

In alternative cases, conservative amino acid changes include changesbased on considerations of hydrophilicity or hydrophobicity, size orvolume, or charge. Amino acids can be generally characterized ashydrophobic or hydrophilic, depending primarily on the properties of theamino acid side chain. A hydrophobic amino acid exhibits ahydrophobicity of greater than zero, and a hydrophilic amino acidexhibits a hydrophilicity of less than zero, based on the normalizedconsensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio.179:125-142, 184). Genetically encoded hydrophobic amino acids includeGly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically encodedhydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, andLys. Non-genetically encoded hydrophobic amino acids includet-butylalanine, while non-genetically encoded hydrophilic amino acidsinclude citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided basedon the characteristics of their side chains. For example, an aromaticamino acid is a hydrophobic amino acid with a side chain containing atleast one aromatic or heteroaromatic ring, which can contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR,etc., where R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,(C₀-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl,substituted (C₀-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₀-C₂₀) aryl,(C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe, Tyr, and Trp.

An non-polar or apolar amino acid is a hydrophobic amino acid with aside chain that is uncharged at physiological pH and which has bonds inwhich a pair of electrons shared in common by two atoms is generallyheld equally by each of the two atoms (i.e., the side chain is notpolar). Genetically encoded apolar amino acids include Gly, Leu, Val,Ile, Ala, and Met. Apolar amino acids can be further subdivided toinclude aliphatic amino acids, which is a hydrophobic amino acid havingan aliphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala, Leu, Val, and Ile.

A polar amino acid is a hydrophilic amino acid with a side chain that isuncharged at physiological pH, but which has one bond in which the pairof electrons shared in common by two atoms is held more closely by oneof the atoms. Genetically encoded polar amino acids include Ser, Thr,Asn, and Gln.

An acidic amino acid is a hydrophilic amino acid with a side chain pKavalue of less than 7. Acidic amino acids typically have negativelycharged side chains at physiological pH due to loss of a hydrogen ion.Genetically encoded acidic amino acids include Asp and Glu. A basicamino acid is a hydrophilic amino acid with a side chain pKa value ofgreater than 7. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include Arg, Lys, and His.

A percent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the comparison window. The “longer” sequenceis the one having the most actual residues in the comparison window(gaps introduced by WU-Blast-2 to maximize the alignment score areignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequence the disclosedpolypeptide, it is understood that in one case, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

In one case, only identities are scored positively (+1) and all forms ofsequence variation including gaps are assigned a value of “0”, whichobviates the need for a weighted scale or parameters as described belowfor sequence similarity calculations. Percent sequence identity can becalculated, for example, by dividing the number of matching identicalresidues by the total number of residues of the “shorter” sequence inthe aligned region and multiplying by 100. The “longer” sequence is theone having the most actual residues in the aligned region.

Scaffolds

As noted herein, the antibodies of the present disclosure can comprise ascaffold structure into which the CDR(s) described above can be grafted.In one embodiment, the scaffold structure is a traditional antibodystructure, that is, an antibody comprising two heavy and two light chainvariable domain sequences. In some cases, the antibody combinationsdescribed herein can include additional components (framework, J and Dregions, constant regions, etc.) that make up a heavy and/or a lightchain. Some embodiments include the use of human scaffold components.

Accordingly, in various embodiments, the antibodies of the disclosurecomprise the scaffolds of traditional antibodies. In some embodiments,the disclosed antibodies can be human and monoclonal antibodies,bispecific antibodies, diabodies, minibodies, domain antibodies,synthetic antibodies, chimeric antibodies, antibody fusions, andfragments of each, respectively. The above described CDRs andcombinations of CDRs can be grafted into any of the following scaffolds.

Chimeric antibodies of the present disclosure can comprise a heavyand/or light chain sequence that is identical or homologous to thecorresponding sequences derived from a particular species. For example,in one embodiment the anti-C5 antibody is a chimeric antibody comprisinga human Fc domain, while the remainder of the antibody can be identicalor homologous to corresponding mouse or rodent sequences. Chimericantibodies can be fragments of such antibodies, so long as the fragmentsexhibit the desired biological activity and comprise sequence that isderived from another species, class of antibody, or subclass of antibody(U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad.Sci. USA 81:6851-6855).

In some embodiments, a variable region of the presently disclosedanti-C5 antibody comprises at least three heavy chain or light chainCDRs, see, supra (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Public Health Service N.I.H., Bethesda, Md.; seealso Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al.,1989, Nature 342: 877-883), embedded within a framework region(designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat etal., 1991, supra; see also Chothia and Lesk, 1987, supra).

In some cases, the antibody can be comprised of a heavy chain variabledomain sequence or a light chain variable domain sequence. In some casesthe heavy or light chain variable domain sequence may comprise asequence selected from the sequences of Table 1.

Traditional antibody structural units, in most cases, comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one light chain (typically having amolecular weight of about 25 kDa) and one heavy chain (typically havinga molecular weight of about 50-70 kDa). The amino-terminal portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant region, whilethe heavy chain may comprise a total of three constant regions (CH1,CH2, and CH3), wherein the constant regions may aid in regulatingeffector function. Human light chains are classified as kappa and lambdalight chains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. IgG has several subclasses, including, but notlimited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including,but not limited to, IgM1 and IgM2.

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about twelve (12) or more amino acids, withthe heavy chain also including a “D” region of about ten (10) more aminoacids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch.7, 2nd ed. Raven Press, N.Y. The variable regions of each light andheavy chain pair form the antibody binding site.

Some naturally occurring antibodies, for example found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter etal., 2001, J. Biol. Chem. 276:26285-26290. Crystallographic studies of acamel antibody have revealed that the CDR3 regions form a surface thatinteracts with the antigen and thus is critical for antigen binding likein the more typical tetrameric antibodies. The disclosure encompassesdimeric antibodies consisting of two heavy chains, or fragments thereof,that can bind to and/or inhibit the biological activity of C5.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three complementarity determining regions or CDRs. The CDRscomprise hypervariable regions of an antibody that are responsible forantigen recognition and binding. The CDRs from the two chains of eachpair are aligned and supported by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest. Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia etal., 1989, Nature 342:878-883.

CDRs constitute the major surface contact points for antigen binding.See, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Further,CDR3 of the light chain and, especially, CDR3 of the heavy chain mayconstitute the most important determinants in antigen binding within thelight and heavy chain variable regions. See, e.g., Chothia and Lesk,1987, supra; Desiderio et al., 2001, J. Mol. Biol. 310:603-615; Xu andDavis, 2000, Immunity 13:37-45; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290; and Muyldermans, 2001, J. Biotechnol. 74:277-302. Insome antibodies, the heavy chain CDR3 appears to constitute the majorarea of contact between the antigen and the antibody. Desmyter et al.,2001, supra. In vitro selection schemes in which CDR3 alone is variedcan be used to vary the binding properties of an antibody. Muyldermans,2001, supra; Desiderio et al., 2001, supra.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is not present in the mature antibody. Apolynucleotide encoding an antibody of the disclosure may encode anaturally occurring signal sequence or a heterologous signal sequence asdescribed below.

In one embodiment, the anti-C5 antibody is a monoclonal antibody, withfrom one (1) to six (6) of the CDRs, as outlined herein. The antibodiesof the disclosure can be of any type including IgM, IgG (including IgG1,IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In some embodiments, theantibody is an IgG type antibody. In one embodiment, the antibody is anIgG2 type antibody.

In some embodiments, the antibody can comprise complete heavy and lightchains, where the CDRs are all from the same species, e.g., human.Alternatively, for example in embodiments wherein the antibody containsless than six CDRs from the sequences outlined above, additional CDRscan be either from other species (e.g., murine CDRs), or can bedifferent human CDRs than those depicted in the sequences. For example,human HC CDR3 and LC CDR3 regions from the appropriate sequencesidentified herein can be used, with HC CDR1, HC CDR2, LC CDR1 and LCCDR2 being optionally selected from alternate species, or differenthuman antibody sequences, or combinations thereof. For example, the CDRsof the disclosure can replace the CDR regions of commercially relevantchimeric or humanized antibodies.

Specific embodiments can include scaffolds of the antibodies thatcomprise human sequences.

In some embodiments, however, the scaffold components can be a mixturefrom different species. As such, the antibody can be a chimeric antibodyand/or a humanized antibody. In general, both chimeric antibodies andhumanized antibodies can be antibodies that combine regions or aminoacids from more than one species. For example, chimeric antibodies, inmost embodiments, comprise variable region(s) from a mouse, rat, rabbit,or other suitable non-human animal, and the constant region(s) from ahuman. In other embodiments, chimeric antibodies comprise human FRsequences and non-human CDRs.

Humanized antibodies are antibodies that are originally derived fromnon-human antibodies, for example a mouse antibody. In variousembodiments of a humanized anti-C5 antibody, the variable-domainframework regions or framework amino acids, which are derived from anon-human antibody, can be changed to amino acid identities found atcorresponding positions in human antibodies. In some embodiments of ahumanized antibody, the entire antibody, except the CDRs, can be encodedby a polynucleotide of human origin or can be identical to such anantibody except within its CDRs. In other embodiments, a humanizedantibody may comprise specific amino acid positions whose identity hasbeen changed to the identity of the same or similar position in acorresponding human antibody. The CDRs, some or all of which can beencoded by nucleic acids originating in a non-human organism, aregrafted into the beta-sheet framework of a human antibody variableregion to create an antibody, the specificity of which is determined bythe engrafted CDRs. The creation of such antibodies is described in,e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al.,1988, Science 239:1534-1536. Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. Roque et al.,2004, Biotechnol. Prog. 20:639-654. In some embodiments, the CDRs can behuman, and thus both humanized and chimeric antibodies, in this context,can include some non-human CDRs. In some cases, humanized antibodies canbe generated that comprise the HC CDR3 and LC CDR3 regions, with one ormore of the other CDR regions being of a different special origin.

In one embodiment, the C5 antibody can be a multispecific antibody, andnotably a bispecific antibody, (e.g. diabodies). These are antibodiesthat bind to two (or more) different antigens, for example C5, andanother antigen, or two different epitopes of C5. Diabodies can bemanufactured in a variety of ways known in the art (Holliger and Winter,1993, Current Opinion Biotechnol. 4:446-449), e.g., prepared chemicallyor from hybrid hybridomas.

In one embodiment, the anti-C5 antibody is a minibody. Minibodies areminimized antibody-like proteins comprising a scFv joined to a CH3domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

In one embodiment, the anti-C5 antibody is a domain antibody; see, forexample U.S. Pat. No. 6,248,516. Domain antibodies (dAbs) are functionalbinding domains of antibodies, corresponding to the variable regions ofeither the heavy (VH) or light (VL) chains of human antibodies dABs havea molecular weight of approximately 13 kDa, or less than one-tenth thesize of a full antibody. dABs are well expressed in a variety of hostsincluding bacterial, yeast, and mammalian cell systems. In addition,dAbs are highly stable and retain activity even after being subjected toharsh conditions, such as freeze-drying or heat denaturation. See, forexample, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; USSerial No. 2004/0110941; European Patent 0368684; U.S. Pat. No.6,696,245, WO04/058821, WO04/003019 and WO03/002609, all incorporatedentirely by reference.

In one embodiment, the anti-C5 antibody is an antibody fragment, that isa fragment of any of the antibodies outlined herein that retain bindingspecificity to C5. In various embodiments, the antibodies are a F(ab),F(ab′), F(ab′)2, Fv, or a single chain Fv fragments. At a minimum, anantibody, as meant herein, comprises a polypeptide that can bindspecifically to an antigen, wherein the polypeptide comprises all orpart of a light and/or a heavy chain variable region.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546) which consists of a singlevariable, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) diabodies ortriabodies, multivalent or multispecific fragments constructed by genefusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448). The antibody fragments can be modified. For example, themolecules can be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245).

In one embodiment, the C5 antibody is a traditional antibody, forexample a human immunoglobulin. In this embodiment, as outlined above,specific structures comprise complete heavy and light chains depictedcomprising the CDR regions. Additional embodiments utilize one or moreof the CDRs of the disclosure, with the other CDRs, framework regions, Jand D regions, constant regions, etc., coming from other humanantibodies. For example, the CDRs of the disclosure can replace the CDRsof any number of human antibodies, particularly commercially relevantantibodies.

In one embodiment, the C5 antibody is an antibody fusion protein (e.g.an antibody conjugate). In this embodiment, the antibody is fused to aconjugation partner. The conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody (see the discussion on covalent modifications ofthe antibodies) and on the conjugate partner. For example linkers areknown in the art; for example, homo- or hetero-bifunctional linkers asare well known (see, Pierce Chemical Company catalog, technical sectionon cross-linkers, pages 155-200, incorporated herein by reference).

In one embodiment, the C5 antibody is an antibody analog. In some casesantibody analogs can be referred to as synthetic antibodies. Forexample, a variety of recent work utilizes either alternative proteinscaffolds or artificial scaffolds with grafted CDRs. Such scaffoldsinclude, but are not limited to, mutations introduced to stabilize thethree-dimensional structure of the antibody as well as wholly syntheticscaffolds consisting for example of biocompatible polymers. See, forexample, Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(PAMs) can be used, as well as work based on antibody mimetics utilizingfibronectin components as a scaffold.

VH and VL Variants

As outlined above, in some embodiments the disclosure providesantibodies comprising, or consisting of a heavy chain variable regioncomprising SEQ ID NO:2, 4, 6, 8, 10, and 12 and/or a light chainvariable region of SEQ ID NO:1, 3, 5, 7, 9, and 11, respectively, orfragments thereof as defined above. Thus, in those embodiments, theantibody comprises not only at least one CDR or variant, but also atleast part of a depicted framework sequence. In addition, the disclosureencompasses variants of such heavy chain variable sequences or lightchain variable sequences.

A variant variable region, generally shares an amino acid homology,similarity, or identity of at least 80% with those of a parent variableregion, such as those disclosed herein. In some embodiments, the variantand parent sequence homologies or identities are at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and almost 100%.Nucleic acid sequence homology, similarity, or identity between thenucleotide sequences encoding individual variant VHs and VLs and thenucleic acid sequences depicted herein are at least 70% with thosedepicted herein, and more alternatively with increasing homologies oridentities of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% and almost 100%. In addition, a variant variable region can, inmany embodiments, shares the biological function, including, but notlimited to, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%of the specificity and/or activity of the parent CDR. In some case,homology and/or identity is only measured outside the CDR sequences,which can be identical. In other cases, the homology and/or identity ismeasured throughout the entire sequence, including CDR sequences. Insome embodiments, constant region variants may also be included.

In various cases, homology of amino acid sequences can reflect thepercentage of identity or positives when optimally aligned as describedabove. In various cases, the % homology (% positive) or % identity canbe calculated by dividing the number of aligned amino acids within acomparison window. A comparison window can be the entire length of oneor the other compared polypeptides, if the two polypeptides are ofunequal length. In other cases, the comparison window can be a portionof one of the polypeptides. In various cases the comparison window formeasuring homology or identity of two polypeptide sequences is greaterthan about 40 aa (amino acids), 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70aa, 75 aa, 80 aa, 85 aa, 90 aa, 95 aa, 100 aa, 150 aa, or 200 aa, and/orless than about 200 aa, 150 aa, 100 aa, 95 aa, 90 aa, 85 aa, 80 aa, 75aa, 70 aa, 65 aa, 60 aa, 55 aa, 50 aa, or 45 aa. In some embodiments, asin the case with various CDR sequences of the present disclosure, thecomparison window may be less than 40 aa, for example between less thanabout 25 aa, 24 aa, 23 aa, 22 aa, 21 aa, 20 aa, 19 aa, 18 aa, 17 aa, 16aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 aa, 9 aa, 8 aa, 7 aa, 6 aa, 5aa, or 4 aa, and greater than about 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa,9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19aa, 20 aa, 21 aa, 22 aa, 23 aa, or 24 aa.

In various cases, the claimed amino acid sequences can have % identityor % homology (% positive) over a given comparison window, that isgreater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% and/or less than about 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, or 75%.

Covalent Modifications of Anti-C5 Antibodies

Covalent modifications of antibodies are included within the scope ofthis disclosure, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction canbe performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues can be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 (all incorporated entirely by reference) are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisdisclosure.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Glycosylation

Another type of covalent modification of the antibodies included withinthe scope of this disclosure comprises altering the glycosylationpattern of the protein. As is known in the art, glycosylation patternscan depend on both the sequence of the protein (e.g., the presence orabsence of particular glycosylation amino acid residues, discussedbelow), or the host cell or organism in which the protein is produced.Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the disclosed antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antibody's amino acid sequence is altered through changesat the DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) can be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antibody can beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites can be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, 1 Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

PEGylation

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Inaddition, as is known in the art, amino acid substitutions can be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG.

Labels

In some embodiments, the covalent modification of the antibodies of thedisclosure comprises the addition of one or more labels.

The term “labelling group” means any detectable label. Examples ofsuitable labelling groups include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labelling group is coupled to theantibody via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand can be used in performing the present disclosure.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which can beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and can be used in performing the present disclosure.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

A fluorescent label can be any molecule that can be detected via itsinherent fluorescent properties. Suitable fluorescent labels include,but are not limited to, fluorescein, rhodamine, tetramethylrhodamine,eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS,BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, theAlexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhodamine,and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham LifeScience, Pittsburgh, Pa.). Suitable optical dyes, includingfluorophores, are described in Molecular Probes Handbook by Richard P.Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

Polynucleotides Encoding Anti-C5 Antibodies

In certain aspects, the disclosure provides nucleic acid moleculesencoding the antibodies described herein. In some cases the disclosednucleic acids code for antibodies, variable regions, or CDRs describedherein. Nucleic acids include both DNA and RNA molecules. Nucleic acidscan be either natural, non-natural nucleic acids, nucleic acid analogs,or synthetic nucleic acids. Nucleic acids of the present disclosure aretypically polynucleic acids; that is, polymers of individual nucleotidesthat are covalently joined by phosphodiester bonds. In various cases thenucleotide sequences can be single-stranded, double stranded, or acombination thereof. The nucleotide sequences can further comprise othernon-nucleic acid molecules such as amino acids, and other monomers.

In many embodiments, the coding sequence may be an isolated nucleic acidmolecule. The isolated nucleic acid molecule is identified and separatedfrom at least one component with which it is ordinarily associated inthe natural source. In some cases a component can be a nucleotidesequence, protein, or non-proteinaceous molecule. An isolated anti-C5antibody-encoding nucleic acid molecule is other than in the form orsetting in which it is found in nature. Isolated anti-C5antibody-encoding nucleic acid molecules therefore are distinguishedfrom the encoding nucleic acid molecule(s) as they exist in naturalcells. However, an isolated anti-C5 antibody-encoding nucleic acidmolecule includes anti-C5 antibody-encoding nucleic acid moleculescontained in cells that ordinarily express anti-C5 antibody where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells. Isolated nucleic acid moleculestherefore are distinguished from the nucleic acid molecule as it existsin an organism. However, in some cases an isolated nucleic acid moleculecan be a nucleic acid contained within a cell, for example, wherein theisolated nucleic acid molecule is introduced into a cell and resides ineither an extrachromosomal location or in a chromosomal locationdifferent from its native location.

Depending on its use, the nucleic acid can be double stranded, singlestranded, or contain portions of both double stranded or single strandedsequence. As will be appreciated by those in the art, the depiction of asingle strand (sometimes referred to as the “Watson” strand) alsodefines the sequence of the other strand (sometimes referred to as the“Crick” strand). A recombinant nucleic can be a nucleic acid, originallyformed in vitro, in general, by the manipulation of nucleic acid byendonucleases, in a form not normally found in nature. Thus an isolatedantibody can be encoded by a nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis disclosure. It is understood that once a recombinant nucleic acid,with all necessary control elements, is made and reintroduced into ahost cell or organism, it can replicate non-recombinantly, i.e., usingthe in vivo cellular machinery of the host cell rather than in vitromanipulations; however, such nucleic acids, once produced recombinantly,although subsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the disclosure.

In some embodiments, the recombinant nucleic acid may comprise one ormore control elements or control sequences. Control element and controlsequence refers to nucleic acid sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers. As used herein, an operablylinked sequence, is a nucleic acid sequence in a functional relationshipwith another nucleic acid sequence. For example, nucleic acid codingsequences can be operably linked to nucleic acid control sequences. Forexample, DNA for a presequence or secretory leader can be operablylinked to DNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation. Inmost embodiments, an operably linked sequence is a DNA sequencecovalently linked to, for example, a secretory leader sequence. However,as described above, some control sequences can be active as RNAsequence. In many embodiments, enhancer sequences are not required to beadjacent to a coding sequence, rather the two sequences may be separatedby one or more nucleic acids.

In various cases, the nucleic acids of the disclosed nucleotidesequences can include nucleotides that are metabolized in a mannersimilar to naturally occurring nucleotides. Also included arenucleic-acid-like structures with synthetic backbone analoguesincluding, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs)(see, e.g.: “Oligonucleotides and Analogues, a Practical Approach,”edited by F. Eckstein, IRL Press at Oxford University Press (1991);“Antisense Strategies,” Annals of the New York Academy of Sciences,Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J.Med. Chem. 36:1923-1937; and “Antisense Research and Applications”(1993, CRC Press)). PNAs contain non-ionic backbones, such asN-(2-aminoethyl) glycine units. Phosphorothioate linkages are describedin: WO 97/03211; WO 96/39154; and Mata (1997) Toxicol. Appl. Pharmacol.144:189-197. Other synthetic backbones encompassed by this term includemethyl-phosphonate linkages or alternating methyl-phosphonate andphosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzyl-phosphonate linkages (Samstag (1996) AntisenseNucleic Acid Drug Dev 6: 153-156).

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids can be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antibody) of the present disclosure. Thus, havingidentified a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids, by simply modifyingthe sequence of one or more codons in a way which does not change theamino acid sequence of the encoded protein.

In various cases, nucleotide sequences encoding the polypeptidesequences of SEQ ID NOS:1-48 are included. These nucleotide codingsequences can be translated into a polypeptide having an amino acidsequence identical to the disclosed polypeptide sequence. In many cases,nucleotides coding for identical polypeptides, may not have identicalnucleotide sequences. The disclosed coding sequences can furthercomprise untranslated sequences, for example poly-adenylation sequences.The inventive coding sequences can also comprise intron or intervening,non-translated, sequence that are spliced out of a transcribed mRNAprior to translation. In various cases the transcribed mRNA can becapped with a terminal 7-methylguanosine. In some embodiments, thecoding sequences will include coding sequences for amino acids that donot appear in the final antibody, for example sequences required forexport of the antibody.

The nucleotide coding sequences can be aligned by BLASTn, as describedabove. In various cases the homology (or identities in BLASTn) of thesealigned nucleotide sequences can be greater than about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and/or less than about100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45%. Invarious cases, the homologous aligned sequences can be less than about700 nt, 600 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 90 nt, 80 nt, 70nt, 60 nt, 50 nt or 40 nt, and/or more than about 50 nt, 60 nt, 70 nt,80 nt, 90 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, or 600 nt.

In various cases, the coding sequence directs transcription of aribonucleic acid sequence that can be translated into amino acidsequence according to the standard genetic code. In various cases, thecode can include variations to the canonical code. In some variations,the coding sequence can include introns, or intervening sequences thatdo not code for amino acids, but can be transcribed and later removedbefore the ribonucleic acid is translated into a polypeptide.

Methods of Producing Antibodies

The present disclosure also provides expression systems and constructsin the form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the disclosure provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas flanking sequences in certain embodiments will typically include oneor more of the following nucleotide sequences: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the C5 antibodycoding sequence; the oligonucleotide sequence can encode a polyHis tag(such as hexaHis), or another “tag” such as FLAG, HA (hemaglutinininfluenza virus), or myc, for which commercially available antibodiesexist. This tag is typically fused to the polypeptide upon expression ofthe polypeptide, and can serve as a means for affinity purification ordetection of the C5 antibody from the host cell. Affinity purificationcan be accomplished, for example, by column chromatography usingantibodies against the tag as an affinity matrix. Optionally, the tagcan subsequently be removed from the purified anti-C5 antibody byvarious means such as using certain peptidases for cleavage.

Flanking sequences can be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence can be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this disclosure can beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence can be known. Here, the flanking sequence can be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it canbe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence can be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationcan be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one can be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes can be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantibody that binds to a C5 polypeptide or C5 epitope. As a result,increased quantities of a polypeptide such as a anti-C5 antibody aresynthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the disclosure will typically containa promoter that is recognized by the host organism and operably linkedto the molecule encoding the C5 antibody. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe gene towhich they are operably linked, that is, with little or no control overgene expression. A large number of promoters, recognized by a variety ofpotential host cells, are well known. A suitable promoter is operablylinked to the DNA encoding heavy chain or light chain comprising a C5antibody of the disclosure by removing the promoter from the source DNAby restriction enzyme digestion and inserting the desired promotersequence into the vector.

In some embodiments, yeast cells may be used to produce the presentlydisclosed anti-C5 antibodies. Suitable promoters for use with yeasthosts are also well known in the art. Yeast enhancers are advantageouslyused with yeast promoters. Suitable promoters for use with mammalianhost cells are well known and include, but are not limited to, thoseobtained from the genomes of viruses such as polyoma virus, fowlpoxvirus, adenovirus (such as Adenovirus 2), bovine papilloma virus, aviansarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and orSimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which can be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence can be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising a C5antibody of the disclosure by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 bp in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer can be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

Expression vectors, for expressing the presently claimed antibodies ofthe disclosure can be constructed from a starting vector such as acommercially available vector. Such vectors may or may not contain allof the desired flanking sequences. Where one or more of the flankingsequences described herein are not already present in the vector, theycan be individually obtained and ligated into the vector. Methods usedfor obtaining each of the flanking sequences are well known to oneskilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding a light chain, a heavy chain, or a light chain and a heavychain comprising an anti-C5 antibody coding sequence has been insertedinto the proper site of the vector, the completed vector can be insertedinto a suitable host cell for amplification and/or polypeptideexpression. The transformation of an expression vector for an anti-C5antibody into a selected host cell can be accomplished by well knownmethods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes ananti-C5 antibody that can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule. A host cell can beeukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines can be selected throughdetermining which cell lines have high expression levels andconstitutively produce antibodies with C5 binding properties. In anotherembodiment, a cell line from the B cell lineage that does not make itsown antibody but has a capacity to make and secrete a heterologousantibody can be selected.

Use of Anti-C5 Antibodies for Diagnostic and Therapeutic Purposes

Antibodies of the disclosure are useful for detecting C5 and/or C5b inbiological samples and identification of cells or tissues that produceC5 protein. In some embodiments, the anti-C5 antibodies of thedisclosure can be used in diagnostic assays, e.g., binding assays todetect and/or quantify C5 expressed in a tissue or cell or C5b in aserum or tissue, or on a cell.

In some embodiments, the antibodies of the disclosure that specificallybind to C5 can be used in treatment of Complement or C5-mediateddiseases in a patient in need thereof. In addition, the anti-C5 antibodyof the disclosure can be used to inhibit C5 from forming a complex withother complement proteins, thereby modulating the biological activity ofC5 in a cell or tissue. Antibodies that bind to C5 thus can modulateand/or block interaction with other binding compounds and as such mayhave therapeutic use in ameliorating Complement and C5 mediateddiseases.

In some embodiments, the binding of C5 by anti-C5 antibodies may resultin disruption of the C5-mediated complement cascade.

Diagnostic Methods

The antibodies of the disclosure can be used for diagnostic purposes todetect, diagnose, or monitor diseases and/or conditions associated withcomplement or C5. The disclosure provides for the detection of thepresence of C5 in a sample using classical immunohistological methodsknown to those of skill in the art (e.g., Tijssen, 1993, Practice andTheory of Enzyme Immunoassays, vol 15 (Eds R. H. Burdon and P. H. vanKnippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al.,1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell Biol.105:3087-3096). The detection of C5 can be performed in vivo or invitro.

Diagnostic applications provided herein include use of the antibodies todetect expression of C5. Examples of methods useful in the detection ofthe presence of C5 include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the antibody typically can be labeled witha detectable labeling group. Suitable labeling groups include, but arenot limited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent groups, biotinyl groups, or predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and can be used in performing the present disclosure.

One aspect of the disclosure provides for identifying a cell or cellsthat express C5. In a specific embodiment, the antibody is labeled witha labeling group and the binding of the labeled antibody to C5 isdetected. In a further specific embodiment, the binding of the antibodyto C5 can be detected in vivo. In a further specific embodiment, theantibody/C5 complex is isolated and measured using techniques known inthe art. See, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodicsupplements); John E. Coligan, ed., 1993, Current Protocols InImmunology New York: John Wiley & Sons.

Another aspect of the disclosure provides for detecting the presence ofa test molecule that competes for binding to C5 with the anti-C5antibodies of the disclosure. An example of one such assay would involvedetecting the amount of free antibody in a solution containing an amountof C5 in the presence or absence of the test molecule. An increase inthe amount of free antibody (i.e., the antibody not bound to C5) wouldindicate that the test molecule is capable of competing for C5 bindingwith the anti-C5 antibody. In one embodiment, the antibody is labeledwith a labeling group. Alternatively, the test molecule is labeled andthe amount of free test molecule is monitored in the presence andabsence of an antibody.

Indications

The complement system has been implicated in contributing to severalacute and chronic conditions, including atherosclerosis,ischemia-reperfusion following acute myocardial infarction,Henoch-Schonlein purpura nephritis, immune complex vasculitis,rheumatoid arthritis, arteritis, aneurysm, stroke, cardiomyopathy,hemorrhagic shock, crush injury, multiple organ failure, hypovolemicshock and intestinal ischemia, transplant rejection, cardiac Surgery,PTCA, spontaneous abortion, neuronal injury, spinal cord injury,myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis,multiple sclerosis, Guillain Bane syndrome, Parkinson's disease,Alzheimer's disease, acute respiratory distress syndrome, asthma,chronic obstructive pulmonary disease, transfusion-related acute lunginjury, acute lung injury, Goodpasture's disease, myocardial infarction,post-cardiopulmonary bypass inflammation, cardiopulmonary bypass, septicshock, transplant rejection, xeno transplantation, burn injury, systemiclupus erythematosus, membranous nephritis, Berger's disease, psoriasis,pemphigoid, dermatomyositis, anti-phospholipid syndrome, inflammatorybowel disease, hemodialysis, leukopheresis, plasmapheresis,heparin-induced extracorporeal membrane oxygenation LDL precipitation,extracorporeal membrane oxygenation, and macular degeneration.

Macular degenerative diseases, such as all stages of age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)forms, choroidal neovascularization (CNV), uveitis, diabetic and otherischemia-related retinopathies, and other intraocular neovasculardiseases, such as diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, Central Retinal VeinOcclusion (CRVO), corneal neovascularization, and retinalneovascularization. One group of complement-associated eye conditionsincludes age-related macular degeneration (AMD), including non-exudative(wet) and exudative (dry or atrophic) AMD, choroidal neovascularization(CNV), diabetic retinopathy (DR), and endophthalmitis.

The presently disclosed anti-C5 antibodies can be used in combinationwith one or more cytokines, lymphokines, hematopoietic factor(s), and/oran anti-inflammatory agent.

Treatment of the diseases and disorders recited herein can include theuse of first line drugs for control of pain and inflammation incombination (pretreatment, post-treatment, or concurrent treatment) withtreatment with one or more of the anti-C5 antibodies provided herein. Insome cases the drugs are classified as non-steroidal, anti-inflammatorydrugs (NSAIDs). Secondary treatments include corticosteroids, slowacting antirheumatic drugs (SAARDs), or disease modifying (DM) drugs.Information regarding the following compounds can be found in The MerckManual of Diagnosis and Therapy, Sixteenth Edition, Merck, Sharp & DohmeResearch Laboratories, Merck & Co., Rahway, N.J. (1992) and inPharmaprojects, PJB Publications Ltd.

In a specific embodiment, the present disclosure is directed to the useof an antibody and any of one or more NSAIDs for the treatment of thediseases and disorders recited herein. NSAIDs owe theiranti-inflammatory action, at least in part, to the inhibition ofprostaglandin synthesis (Goodman and Gilman in “The PharmacologicalBasis of Therapeutics,” MacMillan 7th Edition (1985)). NSAIDs can becharacterized into at least nine groups: (1) salicylic acid derivatives;(2) propionic acid derivatives; (3) acetic acid derivatives; (4) fenamicacid derivatives; (5) carboxylic acid derivatives; (6) butyric acidderivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more salicylic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. Such salicylic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: acetaminosalol,aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate, magnesium salicylate,choline salicylate, diflusinal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide O-acetic acid, salsalate, sodium salicylate andsulfasalazine. Structurally related salicylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more propionic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The propionic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: alminoprofen,benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen,flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen,ibuprofen aluminum, ibuproxam, indoprofen, isoprofen, ketoprofen,loxoprofen, miroprofen, naproxen, naproxen sodium, oxaprozin,piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid,pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen. Structurallyrelated propionic acid derivatives having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In yet another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more acetic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The acetic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac potassium, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium,zidometacin and zomepirac. Structurally related acetic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more fenamic acid derivatives,prodrug esters or pharmaceutically acceptable salts thereof. The fenamicacid derivatives, prodrug esters and pharmaceutically acceptable saltsthereof comprise: enfenamic acid, etofenamate, flufenamic acid,isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid,mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamicacid and ufenamate. Structurally related fenamic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more carboxylic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The carboxylic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof which can be used comprise:clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine.Structurally related carboxylic acid derivatives having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

In yet another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more butyric acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The butyric acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more oxicams, prodrug esters,or pharmaceutically acceptable salts thereof. The oxicams, prodrugesters, and pharmaceutically acceptable salts thereof comprise:droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide.Structurally related oxicams having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more pyrazoles, prodrugesters, or pharmaceutically acceptable salts thereof. The pyrazoles,prodrug esters, and pharmaceutically acceptable salts thereof which canbe used comprise: difenamizole and epirizole. Structurally relatedpyrazoles having similar analgesic and anti-inflammatory properties arealso intended to be encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatmentor, concurrent treatment) with any of one or more pyrazolones, prodrugesters, or pharmaceutically acceptable salts thereof. The pyrazolones,prodrug esters and pharmaceutically acceptable salts thereof which canbe used comprise: apazone, azapropazone, benzpiperylon, feprazone,mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone,propylphenazone, ramifenazone, suxibuzone and thiazolinobutazone.Structurally related pyrazalones having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more of the following NSAIDs:ε-acetamidocaproic acid, S-adenosyl-methionine, 3-amino-4-hydroxybutyricacid, amixetrine, anitrazafen, antrafenine, bendazac, bendazac lysinate,benzydamine, beprozin, broperamole, bucolome, bufezolac, ciproquazone,cloximate, dazidamine, deboxamet, detomidine, difenpiramide,difenpyramide, difisalamine, ditazol, emorfazone, fanetizole mesylate,fenflumizole, floctafenine, flumizole, flunixin, fluproquazone,fopirtoline, fosfosal, guaimesal, guaiazolene, isonixirn, lefetamineHCl, leflunomide, lofemizole, lotifazole, lysin clonixinate,meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin,oxaceprol, oxapadol, paranyline, perisoxal, perisoxal citrate, pifoxime,piproxen, pirazolac, pirfenidone, proquazone, proxazole, thielavin B,tiflamizole, timegadine, tolectin, tolpadol, tryptamid and thosedesignated by company code number such as 480156S, AA861, AD1590,AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100,EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851,MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152,SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901(4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770.Structurally related NSAIDs having similar analgesic andanti-inflammatory properties to the NSAIDs are also intended to beencompassed by this group.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatmentor concurrent treatment) with any of one or more corticosteroids,prodrug esters or pharmaceutically acceptable salts thereof for thetreatment of the diseases and disorders recited herein, including acuteand chronic inflammation such as rheumatic diseases, graft versus hostdisease and multiple sclerosis. Corticosteroids, prodrug esters andpharmaceutically acceptable salts thereof include hydrocortisone andcompounds which are derived from hydrocortisone, such as21-acetoxypregnenolone, alclomerasone, algestone, amcinonide,beclomethasone, betamethasone, betamethasone valerate, budesonide,chloroprednisone, clobetasol, clobetasol propionate, clobetasone,clobetasone butyrate, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacon, desonide, desoximerasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flumethasone pivalate,flucinolone acetonide, flunisolide, fluocinonide, fluorocinoloneacetonide, fluocortin butyl, fluocortolone, fluocortolone hexanoate,diflucortolone valerate, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandenolide, formocortal,halcinonide, halometasone, halopredone acetate, hydro-cortamate,hydrocortisone, hydrocortisone acetate, hydro-cortisone butyrate,hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more slow-acting antirheumaticdrugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS),prodrug esters, or pharmaceutically acceptable salts thereof for thetreatment of the diseases and disorders recited herein, including acuteand chronic inflammation such as rheumatic diseases, graft versus hostdisease and multiple sclerosis. SAARDs or DMARDS, prodrug esters andpharmaceutically acceptable salts thereof comprise: allocupreide sodium,auranofin, aurothioglucose, aurothioglycanide, azathioprine, brequinarsodium, bucillamine, calcium 3-aurothio-2-propanol-1-sulfonate,chlorambucil, chloroquine, clobuzarit, cuproxoline, cyclo-phosphamide,cyclosporin, dapsone, 15-deoxyspergualin, diacerein, glucosamine, goldsalts (e.g., cycloquine gold salt, gold sodium thiomalate, gold sodiumthiosulfate), hydroxychloroquine, hydroxychloroquine sulfate,hydroxyurea, kebuzone, levamisole, lobenzarit, melittin,6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil,myoral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such asSKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine.Structurally related SAARDs or DMARDs having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more COX2 inhibitors, prodrugesters or pharmaceutically acceptable salts thereof for the treatment ofthe diseases and disorders recited herein, including acute and chronicinflammation. Examples of COX2 inhibitors, prodrug esters orpharmaceutically acceptable salts thereof include, for example,celecoxib. Structurally related COX2 inhibitors having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group. Examples of COX-2 selective inhibitors include but notlimited to etoricoxib, valdecoxib, celecoxib, licofelone, lumiracoxib,rofecoxib, and the like.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more antimicrobials, prodrugesters or pharmaceutically acceptable salts thereof for the treatment ofthe diseases and disorders recited herein, including acute and chronicinflammation. Antimicrobials include, for example, the broad classes ofpenicillins, cephalosporins and other beta-lactams, aminoglycosides,azoles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides,lincosamides and polymyxins. The penicillins include, but are notlimited to penicillin G, penicillin V, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacillin, floxacillin, ampicillin,ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, hetacillin,cyclacillin, bacampicillin, carbenicillin, carbenicillin indanyl,ticarcillin, ticarcillin/clavulanate, azlocillin, mezlocillin,peperacillin, and mecillinam. The cephalosporins and other beta-lactamsinclude, but are not limited to cephalothin, cephapirin, cephalexin,cephradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan,cefoxitin, ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime,moxalactam, ceftizoxime, cetriaxone, cephoperazone, ceftazidime,imipenem and aztreonam. The aminoglycosides include, but are not limitedto streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycinand neomycin. The azoles include, but are not limited to fluconazole.The quinolones include, but are not limited to nalidixic acid,norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin andtemafloxacin. The macrolides include, but are not limited toerythomycin, spiramycin and azithromycin. The rifamycins include, butare not limited to rifampin. The tetracyclines include, but are notlimited to spicycline, chlortetracycline, clomocycline, demeclocycline,deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,minocycline, oxytetracycline, penimepicycline, pipacycline,rolitetracycline, sancycline, senociclin and tetracycline. Thesulfonamides include, but are not limited to sulfanilamide,sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole andco-trimoxazole (trimethoprim/sulfamethoxazole). The lincosamidesinclude, but are not limited to clindamycin and lincomycin. Thepolymyxins (polypeptides) include, but are not limited to polymyxin Band colistin.

Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Compositions are disclosed comprising a therapeutically effective amountof one or a plurality of the antibodies of the disclosure together witha pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In addition, the disclosure providesmethods of treating a patient by administering such pharmaceuticalcomposition. A patient can be either a human subject or an animalsubject.

Pharmaceutical compositions comprising one or more anti-C5 antibodiescan be used to reduce C5 activity. Pharmaceutical compositionscomprising one or more antibodies can be used in treating theconsequences, symptoms, and/or the pathology associated with C5activity. In various embodiments, pharmaceutical compositions comprisingone or more antibodies can be used in methods of inhibiting thecomplement pathway. Pharmaceutical compositions comprising one or moreantibodies can be used in methods of treating the consequences,symptoms, and/or the pathology associated with C5 activity.Pharmaceutical compositions comprising one or more antibodies can beused in methods of inhibiting MAC production. Pharmaceuticalcompositions comprising one or more antibodies can be used in methods ofinhibiting Macular Degeneration.

Various acceptable formulation materials are nontoxic to recipients atthe dosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of anti-C5 antibodies are provided.

In certain embodiments, acceptable formulation materials are nontoxic torecipients at the dosages and concentrations employed. In certainembodiments, the pharmaceutical composition may contain formulationmaterials for modifying, maintaining or preserving, for example, the pH,osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. In such embodiments, suitable formulation materialsinclude, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates orother organic acids); bulking agents (such as mannitol or glycine);chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides; disaccharides; and other carbohydrates (such asglucose, mannose or dextrins); proteins (such as serum albumin, gelatinor immunoglobulins); coloring, flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, PEG, sorbitan esters,polysorbates such as polysorbate 20, polysorbate, triton, tromethamine,lecithin, cholesterol, tyloxapal); stability enhancing agents (such assucrose or sorbitol); tonicity enhancing agents (such as alkali metalhalides, sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18^(th) Edition, (A. R. Genrmo,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the disclosure. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition can be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier canbe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and mayfurther include sorbitol or a suitable substitute therefor. In certainembodiments of the disclosure, C5 antibody compositions can be preparedfor storage by mixing the selected composition having the desired degreeof purity with optional formulation agents (REMINGTON'S PHARMACEUTICALSCIENCES, supra) in the form of a lyophilized cake or an aqueoussolution. Further, in certain embodiments, the C5 antibody product canbe formulated as a lyophilizate using appropriate excipients such assucrose.

The pharmaceutical compositions of the disclosure can be selected forparenteral delivery. Alternatively, the compositions can be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components can be present in concentrations that areacceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this disclosure can be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired C5 antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the C5 antibody is formulated as a sterile,isotonic solution, properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredantibody.

Pharmaceutical compositions of the disclosure can be formulated forinhalation. In these embodiments, C5 antibodies are advantageouslyformulated as a dry, inhalable powder. In specific embodiments, C5antibody inhalation solutions may also be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions can benebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Application No.PCT/US94/001875, which is incorporated by reference and describespulmonary delivery of chemically modified proteins. It is alsocontemplated that formulations can be administered orally. C5 antibodiesthat are administered in this fashion can be formulated with or withoutcarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the C5 antibody. Diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be employed.

A pharmaceutical composition of the disclosure is provided to comprisean effective quantity of one or a plurality of C5 antibodies in amixture with non-toxic excipients that are suitable for the manufactureof tablets. By dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form.Suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; or binding agents, such as starch, gelatin, oracacia; or lubricating agents such as magnesium stearate, stearic acid,or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving C5 antibodies in sustained-or controlled-delivery formulations. Techniques for formulating avariety of other sustained- or controlled-delivery means, such asliposome carriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art. See, forexample, International Patent Application No. PCT/US93/00829, which isincorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method can beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations canbe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The disclosure alsoprovides kits for producing a single-dose administration unit. The kitsof the disclosure may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this disclosure, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of a C5 antibody-containingpharmaceutical composition to be employed will depend, for example, uponthe therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication forwhich the C5 antibody is being used, the route of administration, andthe size (body weight, body surface or organ size) and/or condition (theage and general health) of the patient. In certain embodiments, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect. A typical dosage may range fromabout 0.1 μg/kg to up to about 30 mg/kg or more, depending on thefactors mentioned above. In specific embodiments, the dosage may rangefrom 0.1 μg/kg up to about 30 mg/kg, optionally from 1 μg/kg up to about30 mg/kg or from 10 μg/kg up to about 5 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular C5 antibody in the formulation used. Typically, a clinicianadministers the composition until a dosage is reached that achieves thedesired effect. The composition may therefore be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of the desired molecule) over time, or as a continuousinfusion via an implantation device or catheter. Further refinement ofthe appropriate dosage is routinely made by those of ordinary skill inthe art and is within the ambit of tasks routinely performed by them.Appropriate dosages can be ascertained through use of appropriatedose-response data. In certain embodiments, the antibodies of thedisclosure can be administered to patients throughout an extended timeperiod. Chronic administration of an antibody of the disclosureminimizes the adverse immune or allergic response commonly associatedwith antibodies that are not fully human, for example an antibody raisedagainst a human antigen in a non-human animal, for example, a non-fullyhuman antibody or non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intravitreal,sub-retinal, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. In certainembodiments, the compositions can be administered by bolus injection orcontinuously by infusion, or by implantation device.

The composition also can be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device can be implanted intoany suitable tissue or organ, and delivery of the desired molecule canbe via diffusion, timed-release bolus, or continuous administration. Forocular implants, the implant can be implanted via intra-ocularinjection, intravitreal injection, sub-retinal injection, suprachoroidalinjection, retrobulbar injection or injection into sub-Tenon space.

It also can be desirable to use C5 antibody pharmaceutical compositionsaccording to the disclosure ex vivo. In such instances, cells, tissuesor organs that have been removed from the patient are exposed to C5antibody pharmaceutical compositions after which the cells, tissuesand/or organs are subsequently implanted back into the patient.

In particular, C5 antibodies can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the C5 antibody. In certainembodiments, such cells can be animal or human cells, and can beautologous, heterologous, or xenogeneic. In certain embodiments, thecells can be immortalized. In other embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulatedto avoid infiltration of surrounding tissues. In further embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

All references cited within the body of the instant specification arehereby expressly incorporated by reference in their entirety.

EXAMPLES

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the disclosure.

Example 1—Immunization and Hybridoma Creation

For generation of hybridomas and monoclonal antibodies, immunization andscreening were conducted essentially as described in Antibodies, Alaboratory Mannual, Cold Spring Harbor Laboratory. The procedurespecific to the generation of anti-C5 monoclonal antibodies as describedin this application is briefly described as follows.B10.D2-Hc^(O)H2^(d)H2-T18^(c)/02SnJ mice deficient in complement C5(Jackson Labs®, Bar Harbor maine), were immunized by foot pad injectionusing 75 μg of human C5 (Quidel® cat #A403) in Complete Freundsadjuvant, followed by sequential secondary boosts by intraperitoneal(I.P.) administration using 75 μg C5 protein with Incomplete Freund'sadjuvant on day 28. ELISA screen for serum titers for reactivity againstC5 protein were conducted 9-10 days post-secondary boost. For theinitial set of fusions mice showing favorable titers were immunized withfusion boosts (75 μg C5 in pBS, I.P.) on day 82, 83 and 84 with spleenfusion into SP2/0 mouse myeloma using standard techniques on day 85. Asecond cohort of mice was further immunized on day 68 and 175 followedby fusion boosts day 195, 196 and 197 with fusion on day 198. All fusionwells were screen for reactivity against C5 protein by ELISA 18 dayspost fusion and positive hybridomas subcloned using standard techniquesallowing derivation of monoclonal antibodies.

Example 2—Hybridoma Culture

The hybridomas were maintained in DMEM containing 15% Fetal Clone II,OPI, HAT, non-essential amino acids and recombinant mouse IL-6.Hybridoma supernatants were screened by enzyme-linked immunosorbentassay (ELISA) to detect antihuman C5 antibodies. Positive cultures forC5 were expanded in DMEM containing 15% Fetal Clone II, OPI andnon-essential amino acids, and subcloned twice by limiting dilution. Thesubcloned hybridomas were isotyped with SBA Clonotyping System/HRP(SouthernBiotech) according to the manufacturer's protocol.

Example 3—Cloning and Sequence Determination of Monoclonal VariableHeavy and Light Chain Domains

Variable light (VL) and heavy (VH) chains domains were cloned followingthe de novo RT-PCR amplification. Briefly, total RNA was isolated fromselected subcloned hybridoma cell lines using a total RNA isolation kit(Qiagen®). cDNA synthesis was performed using the First Strand cDNASynthesis Kit (Invitrogen®). The forward primers were specific for theN-terminal amino acid sequence of the VL and VH region, and the LC andHC reverse primers were designed to anneal to a region in the constantlight (CL) and constant heavy domain 1 (CH1). The primers used for donovo cloning is listed below. Amplified VL or VH fragments were isolatedand subcloned into pCR®II-TOP vector (Invitrogen®, Life Technologies®)and sequenced using standard methods.

TABLE 4 Primers VH Forward  MuIgVH5′ PrimersGGGAATTCATGRASTTSKGGYTMARCTKGRTTTGG SEQ ID GAATTCATGRAATGSASCTGGGTYWTYCTCTTACTAGT NO: 49CGACATGAAGWTGTGGBTRAACTGGRTACTAGTCGACATGGRATGGASCKKIRTCTTTMTCTACTAGTCGACATGAACTTYGGGYTSAGMTTGRTTTACTAGTCGACATGTACTTGGGACTGAGCTGTGTATACTAGTCGACATGAGAGTGCTGATTCTTTTGTGACTAGTCGACATGGATTTTGGGCTG ATTTTTTTTATTG VH Reverse MuIgGVH3′- Primers  CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG SEQ ID NO: 50VL Forward  MuIgKVL5′ primers  GGGAATTCATGRAGWCACAKWCYCAGGTCTTTA SEQ IDCTAGTCGACATGAGIMMKTCIMTTCAITTCYTGGGACT NO: 51AGTCGACATGAKGTHCYCIGCTCAGYTYCTIRGACTAGTCGACATGGTRTCCWCASCTCAGTTCCTTGACTAGTCGACATGTATATATGTTTGTTGTCTATTTCTACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTACTAGTCGACATGGATTTWCARGTGCAGATTWTCAGCTTACTAGTCGACATGGTYCTYATVTCCTTGCTGTTCTGGACTAGTCG ACATGGTYCTYATVTTRCTGCTGCTATGGVL Reverse  MuIgKVL3′ Primers CCCAAGCTTACTGGATGGTGGGAAGATGGA SEQ ID NO: 52

PCR was performed as follows:

cDNA 5 μL10× PCR buffer 5 μLdNTP 1 μLprimer mix 2.5 μL

Polymerase 1 μL

dH2O 35.5 μLTotal volume, 50 μL

TABLE 5 PCR Conditions Step Temp (° C.) Time (min) 1 95 5:00 2 95 0:30 358 0:30 4 72 3:00 5 Return to Step 2, repeat 34 times 6 72 5:00 7  4storage

Example 4—Anti-C5 Inhibitory Activity Screen (CH50 Hemolytic Assay)

Sheep red blood cells (RBC) (innovative research IC100-0210) were primedby incubating anti-RBC stroma antibody (Sigma Aldrich, Cat. No. 58014)for 1 hour at 37° C. followed by washing and resuspension in GVB++buffer at a concentration of 5×10⁸/mL and stored at 4° C. until use. Foranalysis of hemolytic activity, RBCs were diluted to a finalconcentration of 4.1×10⁷/ml in the presence of human serum in GVB++buffer followed by incubation for 1 hour at 37° C. The level ofhemolytic activity was determined by pelleting unlysised RBC andcellular debris at 10,000×g for 10 minutes at 4° C. and measuring levelsof released hemoglobin in the supernatant by monitoring the absorbanceat 541 nm. In studies examining functional activity of antibodies, serumand antibodies were incubated for 20 minutes at 4° C. prior to additionto red blood cells. For testing activity in hybridoma cell culturesupernatants, supernatants were incubated with 3% NHS in GVB buffer at1:1 ratio for 60 minutes at 4° C. prior to the addition of primed RBC.Controls included serum alone (positive control), dH₂O (100% lysis), andSerum+EDTA 10 mM (negative control) For analysis of the alternativepathway GVB+10 mM EGTA (Boston Bioproducts IBB-310) and C1Q deficienthuman serum (Quidel, A509) was used. In some assays unprimed rabbit redblood cells (1×10⁷) are substituted for sheep red blood cells and theassay is run in the presence of GVB buffer containing 0.5 mM EGTA(Boston Bioproducts IBB-310).

FIG. 3 is a graphical representation of the results of the hemolyticassay for a selected number of clones screened. The black line betweenclones 5B201 and 5D7-5 represents results from the commercially purchasemouse monoclonal antibody A239 (Quidel A239). Clones to the left of thisline represents antibodies that showed higher/better inhibition ofcomplement activation (which results in lysis of cells). One subclone ofparticular interest was 10C9 (and progeny, with the nomenclature of10C9-X, with X representing a different subclone number from theparent).

Example 5—Anti-C5 Inhibitory Activity Screen (IgM ELISA Assay)

96-well EIA plates (Costar #3590) were coated with 2 μg/ml human IgM incoating buffer pH 9.5. (BD-biosciences 51-2713KC) overnight at 4° C.Plates were washed using wash buffer (BD-biosciences 51-9003739). Serumdiluted to 2% in GVB (BD-biosciences 51-2713KC) and was combined withvarying concentration of hybridoma supernatant or purified IgGs andincubated for 20 minutes at 4° C. After the incubation period 100 ul ofthe serum/antibody mixture is added to the washed IgM coated plates andincubated for 1 hr at 37° C. After the incubation period plates werewashed three times with wash buffer and then incubated with anti-C5b-9mouse monoclonal antibody (Quidel A239) at a 1:10.000 dilution in assaydiluent (BD-biosciences 51-2641KC) for 30 minutes at room temperature.After incubation plates were washed three times and then probed withgoat anti-mouse HRP conjugate diluted 1:3000 in assay diluent. Plateswere incubated for 30 minutes and the washed three times in wash bufferand the signal detected by the addition of substrate (BD-biosciences51-2606KZ and BD-biosciences 51-2607KZ) followed by incubation at roomtemperature for 10 minutes prior to addition of stop solution(BD-biosciences 51-2608KZ). Level of complement activation was thendetermined by read the absorption at 450 nm.

FIGS. 5A, 5B, and 5C shows the results of the IgM ELISA using wholeserum, in which all complement pathways are active; using C2 deficientserum, where only the alternative pathway is active; and using Factor Bdeficient serum where the classical and lectin pathways are active. TheA239 antibody (Quidel A239) against C5 (labelled Anti-C5 in FIGS. 5A-5C)served as a negative control. An anti-Factor D antibody (labelledAnti-FD in FIGS. 5A-5C) served as a positive control comparator in thealternative pathway (FIG. 5B). Overall, the 10C9-19 antibody performedequally well under all three conditions serum conditions.

Example 6—Anti-C5 ELISA

96-well EIA plates (Costar #3590) are coated with 1 μg/ml Human C5 incoating buffer pH 9.5. (BD-biosciences 51-2713KC) overnight at 4° C.Following day plates were washed Plates are washed using wash buffer(BD-biosciences 51-9003739), and then blocked for 30 minutes using AssayDiluent (BD-biosciences 51-2641KC). Purified monoclonal antibodies orhybridoma supernatants were then diluted in to assay diluent and addedto wells previously coated with C5 and incubated at room temperature for60 minutes. Plates were washed 3 times and the level of bound monoclonaldetected using at mouse HPR conjugated secondary and substrate. Level ofbound antibody was determined by measuring absorbance at 450 nM. FIG. 7is a graphical representation of the binding of C5 using selectedmonoclonal antibodies/hybridoma supernatants.

Example 7—Detection of Insoluble C5b-9 Assay

96-well EIA plates (Costar #3590) are coated with 2 μg/ml Human IgM IgM(V) in coating buffer pH 9.5. (BD-biosciences 51-2713KC) overnight at 4°C. Plates are washed using wash buffer (BD-biosciences 51-9003739).Normal Human Serum was diluted to 2% in GVB (BD-biosciences 51-2713KC)and 100 μl of the Serum/GVB mixture was added to the washed IgM coatedplates and incubated for 1 hr at 37° C. After the incubation periodplates are washed three times with wash buffer and then incubated withanti-C5 monoclonal antibodies diluted in to assay diluent to theconcentrations as indicated in the figure. After incubation plates werewashed three times and then probed for 30 minutes with anti-mouse HRPconjugate secondary diluted 1:3000 in assay diluent followed by washingthree times in wash buffer. Bound antibody was then detected by theaddition of substrate (BD-biosciences 51-2606KZ and BD-biosciences51-2607KZ) followed by incubation at room temperature for 10 minutesprior to addition of stop solution (BD-biosciences 51-2608KZ). Level ofcomplement activation is then determined by read the absorption at 450nm.

FIG. 10 shows a graphical representation of the results. Of themonoclonal antibodies screened, 10C9-19r (r is used to designate thatthe antibody that was used is a recombinant version of the 10C9-19clone) does not bind to insoluble C5b9. This is consistent with thehypothesis that this antibody does not recognize or bind C5 after it hasbeen incorporate to MAC.

Example 8—Detection of Soluble C5b-9

Amine reactive tips (AR2G) (ForteBio®, 18-5092) were used for theimmobilization of antibodies in the OCTET RED 96 (ForteBio®). AR2G tipswere first rehydrated in ddH20 for 10 minutes in the loading tray. Uponinitiation of the OCTET protocol, tips were then transferred to asecondary hydration solution of ddH20 for 60 seconds to make sure thereare no aberrant readings. After rehydration, the tips were activated infreshly mixed 20 mM 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC), 10 mM sulfo-N-hydroxysulfosuccinimide (s-NHS) for300 seconds. Antibodies being bound to the AR2G tips were diluted to 20μg/ml in 10 mM Sodium Acetate, pH 5.0. After the AR2G tips wereactivated, they were placed in the antibody solution for 600 seconds.The tips were then quenched in 1M Ethanolamine, pH 8.5 for 300 seconds.After quenching, the tips were moved into Kinetics Buffer for 120seconds to get a baseline reading. Soluble C5b-9 (CompTech, A127) wasdiluted to 30 μg/ml in Kinetics Buffer (KB). After baseline, theantibody bound tips were placed in the soluble C5b-9 solution for 300seconds to measure association. The tips were finally returned to the KBsolution where baseline was measured and a disassociation step wasmeasured for 600 seconds. The level of deflection from baseline at 300seconds of association was used as an indicator of binding affinity. Allsolutions used were in 200 μl volumes per well in a 96 well flat bottomblack plate (Greiner Bio-One, 655209). The OCTET protocol was run at1000 rpm and 30° C.

Results are shown in FIGS. 11A and 11B. The Quidel A239 antibody(labelled A239 in FIGS. 11A and 11B), serves as a positive control as itbinds to C5b-9 (part of the MAC). From the results, as expected, no/verylittle binding was observed with the 10C9-19r antibody. This isconsistent with the hypothesis that 10C9 (and its progeny/subclones) donot bind to soluble C5b-9.

Example 9—C5a Generation Assay

96-well EIA plates (Costar #3590) were coated with 2 μg/ml Human IgM incoating buffer pH 9.5. (BD-biosciences 51-2713KC) overnight at 4° C.Plates were washed using wash buffer (BD-biosciences 51-9003739). Serumdiluted to 10% in GVB (BD-biosciences 51-2713KC) in the presence orabsence of purified IgGs (anti-C5 antibodies) and incubated for 20minutes at 4° C. After the incubation period 100 μl of theserum/antibody mixture is added to the washed IgM coated plates andincubated for 1 hr at 37° C. After the incubation supernatant wascollected. Levels of C5a in the supernatant were then determined usingMicroVue C5a EIA Kit (Quidel, cat #A021).

FIGS. 6A, 6B, and 6C show the results of the assay. FIG. 6A shows levelsof C5a in the supernatant for selected anti-C5 antibodies that werescreened. The black horizontal line depicts background levels. As seenfrom the graph, some antibodies were better than others in blocking C5aformation. FIG. 6B compares 10C9-19 antibody in C5a formation. As seenin the graph, Ms IgG condition served as a positive control and the “NoCVF” (no Cobra Venom Factor) control served as a no protease negativecontrol. At the 5 μg/ml concentration, another anti-C5 antibody, 8C7-26inhibited C5a formation, but did not inhibit C5a formation at the 0.05ug/ml concentration. However, 10C9-19 does not inhibit C5a formation ateither the 5 μg/ml nor at the 0.05 ug/ml concentration.

Example 10—Statistical Analysis

The following describes how percent inhibition and other statisticalanalysis was performed in the experiments included in this Examplesection.

Hemolytic Assay: % inhibition=1−((T−N)/(P−N))*100

T is test OD (level of hemoglobin released during the assay)

N=negative control OD (hemoglobin release within the assay underconditions in which complement activity has been blocked by addition ofEDTA to 10 mM)

P=positive control OD (hemoglobin release when erythrocytes areincubated in the presence of serum in the absence of an inhibitor, thisrepresents 100% activity).

Z-Factor: Z-factor=1−((3*(Dp−Dn))/(abs(Mp−Mn)))

where Dp is standard deviation of positive control, Dn is standarddeviation of negative control, Mp is mean of positive control, and Mn ismean of negative control.

Curve fit (Graphpad Prism)IC90: Y=Ymin+(Ymax−Ymin)/(1+10^((ECx-X)*m)))

where ECx is log IC90−(1/m)*log(90/(100−90)).

Example 11—Immunization of C5 Deficient Mice

Immunization of C5 deficient mice allowed the generation of hybridomacell culture supernatants that are capable of inhibiting complementmediated red blood cell lysis as determined by the CH50 hemolytic assay.The response by the selected hybridomas was much greater than that seenusing conventional commercially available antibodies indicated by theblack line in FIG. 3.

Expansion and cloning of the primary of the hybridomas with sequentialpurification of IgG allow the analysis of function and efficacy inblocking complement mediated cell lysis by titrating the concentrationof the IgG. A more thorough understanding of the relative efficacy of agive monoclonal to inhibit complement mediated cell lysis is obtained,as shown in FIGS. 4A and 4B.

The functional activity of anti C5 monoclonal antibodies can becharacterized based on efficacy for inhibiting a select complementpathways. Inhibitory antibodies were selected based on the particularpathway in which they inhibit, shown in FIGS. 5A, 5B, and 5C.

Blocking cell lysis can occur by either preventing the assembly of themembrane attack complex or by the blocking of the conversion of C5 toC5b by the C5 convertase. Further characterization allows one to examinethe mechanism of inhibition, i.e. if the inhibitory reagent disrupts theproteolytic cleavage of C5 leading to generation of C5b and assembly ofC5b-9 complex or only block assemblage of the C5b-9 complex withoutblocking generation of C5a. In the latter case identification ofinhibitors blocking convertase activity was identified by examining thegeneration of C5a which is an obligatory by product in the production ofC5b. This was done by examining single point determinations or bytitration of the antibody, shown in FIGS. 6A, 6B and 6B.

Specificity of a monoclonal antibody for C5 was identified by examiningits interaction dose dependent interaction with C5 directly coated on toan ELISA plates, shown in FIG. 7.

Further characterization can occur by studying the affinity ofmonoclonal antibodies using Bio-layer interferometry (BLI) allowingidentification of KD values and relative specificity of the monoclonalantibodies, shown in FIG. 8. Additional characterization was obtained bystudying the binding of the monoclonal antibodies to C5 protein insolution, shown in FIG. 9.

Example 12—Selection of C5 Antibodies

One preferred embodiment is the selection for antibodies that do notrecognize C5 once it is incorporated into the membrane attack complex.Monoclonal antibodies were examined according to the ability torecognize C5 with in the C5b-9 complex when deposited into the bottom ofELISA plates after complement activation with IgM, shown in FIG. 10.

Further cross reactivity with C5 within C5b-9 was identified byexamining the ability of the monoclonal to bind soluble C5b-9 usingBio-layer interferometry (BLI) and determining the level of deflection,shown in FIG. 11.

Example 13—Generation of Humanized Antibodies

A lead antibody was selected and humanized. The humanization method ofstring content optimization (Lazar et al, U.S. Pat. No. 7,657,380B2,issued Feb. 2, 2010; U.S. Pat. No. 7,930,107B2, issued Apr. 19, 2011;US20060008883A1, filed Dec. 3, 2004; US20080167449A1, filed Oct. 31,2007; US20110236969A1, filed Mar. 21, 2011; US20100190247A1, filed Mar.12, 2012, all incorporated entirely by reference) was applied to themurine 10C9 antibody. Selected humanized sequences are listed in SEQ IDNOs:1-12 and Table 2.

Using standard techniques, the IgGs were produced. Percent inhibition isshown in FIGS. 12A, 12B and 12C for full-length antibodies using theELISA assay that was described in Example 6 above. Additionally, Fabfragments were produced using standard techniques and their activitysimilar to that of the parent molecule, using the ELISA assay that wasdescribed in Example 6. Graphical representation of the results is shownin FIGS. 13A, 13B, and 13C.

Example 14—Use of C5 Antibody for Prevention of C5b-9 Deposition inRetinal and Choroidal Tissues

Additionally, the therapeutic potential of a compound by intravitrealdelivery in blocking C5b-9 formation in retinal and choroidal tissuescan be assessed by use of standard models leading to complementactivation in tissues of interest as provided in, AL-78898A InhibitsComplement Deposition in a Primate Light Damage Model, ARVO Ab A3872012. Humanized H5L2 (SEQ ID NO:10 and SEQ ID NO: 2, respectively)antibody was humanized from the mouse monoclonal antibody subclone 10C9.H5L2 was tested in a non-human primate light injury model. Intravitrealdosing of the H5L2 antibody provided efficacy in blocking complementdeposition in the retina that was comparable to the negative control(PBS, no light injury, labeled “PBS No BL”). Positive control of PBSwith light injury (labeled “PBS”) was also used. Graphicalrepresentation of the results is shown in FIGS. 14A (retina) and 14B(choroid). These data indicate that local delivery of the H5L2 antibodyis efficacious in an in vivo model relevant to the treatment of maculardegeneration and other ocular indications.

We claim:
 1. A pharmaceutical composition comprising an anti-C5 antibodyand a pharmaceutically acceptable carrier, wherein the anti-C5 antibodybinds to C5 and inhibits complement dependent hemolysis, but does notblock C5a formation.
 2. The composition of claim 1, wherein the antibodyblocks C5 binding to human Complement Component 6 and/or
 7. 3. Thecomposition of claim 1, wherein the antibody inhibits formation ofmembrane attack complex (MAC).
 4. The composition of claim 1, whereinthe antibody comprises a first amino acid sequence and a second aminoacid sequence, wherein: (a) the first sequence comprising: (i) a CDR1selected from the group consisting of SEQ ID NOs:13, 19, 25, 31, 37 and43; (ii) a CDR2 selected from the group consisting of amino acidssequence GTS (SEQ ID NOS: 14 and 20), SGS (SEQ ID NO: 26), RTS (SEQ IDNO: 32), YTS (SEQ ID NO: 38), and WAS (SEQ ID NO: 44); and (iii) a CDR3selected from the group consisting of SEQ ID NOs:15, 21, 27, 33, 39 and45; and (b) the second sequence comprising: (i) a CDR1 selected from thegroup consisting of SEQ ID NOs:16, 22, 28, 34, 40 and 46; (ii) a CDR2selected from the group consisting of SEQ ID NOs:17, 23, 29, 35, 41 and47; and (iii) a CDR3 selected from the group consisting of SEQ IDNOs:18, 24, 30, 36, 42 and
 48. 5. The composition of claim 1, whereinthe antibody further comprises a heavy chain and a light chain wherein:the light chain comprises an amino acid sequence at least 80% identicalto a sequence selected from the group consisting of SEQ ID NO:1, 3, 5,7, 9, and 11; and the heavy chain comprises an amino acid sequence atleast 80% identical to a sequence selected from the group consisting ofSEQ ID NO:2, 4, 6, 8, 10, and 12
 6. The composition of claim 1, whereinthe antibody comprises a light chain and a heavy chain variable domainselected from the light chain and heavy chain variable sequences: SEQ IDNO:1/SEQ ID NO:2; SEQ ID NO:3/SEQ ID NO:4; SEQ ID NO:5/SEQ ID NO:6; SEQID NO:7/SEQ ID NO:8; SEQ ID NO:9/SEQ ID NO:10; SEQ ID NO:11/SEQ IDNO:12; and SEQ ID NO:3/SEQ ID NO:10.
 7. The composition of claim 1,wherein the antibody is a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof.
 8. Thecomposition of claim 7, wherein the antibody is an antibody fragment andthe antibody fragment is a Fab fragment, a Fab′ fragment, a F(ab′)2fragment, a Fv fragment, a diabody, or a single chain antibody molecule.9. The composition of claim 7, wherein said antibody is of the lgG1-,lgG2- IgG3- or IgG4-type.
 10. The composition of claim 9, wherein theantibody is of an IgG1-type.
 11. The composition of claim 1, furthercomprising an additional active agent.
 12. A method for treating orreducing the occurrence of an indication associated with complementactivation in a patient in need thereof comprising administering to saidpatient the pharmaceutical composition of claim 1; and thereby treatingor reducing the occurrence of the indication.
 13. A nucleic acidmolecule encoding an anti-C5 antibody, wherein the antibody binds to C5and inhibits complement dependent hemolysis, but does not block C5aformation, wherein the antibody comprises a first amino acid sequenceand a second amino acid sequence, further wherein: (a) the firstsequence comprising: (i) a CDR1 selected from the group consisting ofSEQ ID NOs:13, 19, 25, 31, 37 and 43; (ii) a CDR2 selected from thegroup consisting of amino acids sequence GTS (SEQ ID NOS: 14 and 20),SGS (SEQ ID NO: 26), RTS (SEQ ID NO: 32), YTS (SEQ ID NO: 38), and WAS(SEQ ID NO: 44); and (iii) a CDR3 selected from the group consisting ofSEQ ID NOs:15, 21, 27, 33, 39 and 45; and (b) the second sequencecomprising: (i) a CDR1 selected from the group consisting of SEQ IDNOs:16, 22, 28, 34, 40 and 46; (ii) a CDR2 selected from the groupconsisting of SEQ ID NOs:17, 23, 29, 35, 41 and 47; and (iii) a CDR3selected from the group consisting of SEQ ID NOs:18, 24, 30, 36, 42 and48.
 14. The nucleic acid molecule according to claim 13, wherein thenucleic acid molecule is operably linked to a control sequence.